Staufen1 regulating agents and associated methods

ABSTRACT

Methods of minimizing dysregulation of Staufen1-associated RNA metabolism can include introducing an amount of a Staufen1-regulating agent to a target cell sufficient to minimize the dysregulation. Therapeutic compositions for treating a neurodegenerative condition associated with Staufen1-induced dysregulation of RNA metabolism can include a therapeutically effective amount of a Staufen1-regulating agent and a pharmaceutically acceptable carrier.

PRIORITY DATA

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/685,269, filed on Jun. 14, 2018, which isincorporated herein by reference.

BACKGROUND

Neurodegenerative diseases occur when nerve cells in the brain orperipheral nervous system lose function over time and ultimately die.Further, nerve cells generally don't reproduce or replace themselves.The risk of being affected by a neurodegenerative disease increases withage. Although treatments may help relieve some of the physical or mentalsymptoms associated with neurodegenerative diseases, there is currentlyno cure. Non-limiting examples of neurodegenerative diseases includeperipheral neuropathy, Alzheimer's disease (AD), Parkinson's disease(PD), Huntington's disease (HD), spinocerebellar ataxia (SCA), priondisease, motor neuron disease (MND), amyotrophic lateral sclerosis(ALS), multiple sclerosis (MS), and spinal muscular atrophy (SMA) amongothers.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and advantage of the presentinvention, reference is made to the following detailed description ofpreferred embodiments and in connection with the accompanying drawings,in which:

FIG. 1A presents images where ATXN2 and Staufen1 co-localize in stressgranules under stress condition. HEK-293 cells were treated with heatshock at 43.5° C. for 1 hr. Fixed cells were immunostained with ATXN2,Staufen1 and TIA-1 (stress granule marker) antibodies. Representativecells showing co-localization of endogenous ATXN2 (red) and Staufen1(green) or ATXN2 (green) and TIA-1 (red) as aggregates by merge images(yellow signals). ATXN2 and Staufen1 co-localizations are not seen undernon-stress (37° C.) condition.

FIG. 1B presents images showing co-localization of ATXN2 and Staufen1 instress-granule-like aggregates in SCA2-derived skin fibroblasts. Normaland SCA2 fibroblasts were immunostained with anti-ATXN2 (green) andanti-Staufen1 (red) antibodies. Aggregation of ATXN2 (green) andStaufen1 (red) signals are seen in the cytoplasm of SCA2 fibroblasts.Merged images of green and red signals are observed as yellow signalsdemonstrate that ATXN2 co-localizes with Staufen1.

FIG. 1C is a graph of the number of aggregates per cell in normal vs.SCA2 fibroblasts.

FIG. 1D presents images showing the increment of granules in SCA2fibroblast under stress condition. Normal and SCA2 fibroblasts wereimmunostained with ATXN2 and Staufen1 antibodies after heat shock at43.5° C. for 1 hr. Representative cells showing granules of endogenousATXN2 (red) and Staufen1 (green) as aggregates by merge images (yellowsignals). Nuclei were stained with DAPI.

FIG. 1E is a graph of the number of granules per cell in normal vs. SCA2fibroblasts.

FIG. 1F presents representative blots of three independent experimentsshowing that Staufen1 interacts with ATXN2 in vivo. Non-RNase or RNasetreated HEK-293 whole cell extracts expressing Flag-tagged ATXN2-(Q22 orQ108) were subjected to immunoprecipitation with Flag mAb beads. Boundprotein complexes were eluted by Flag peptide competition and analyzedby western blot. Staufen1 shows RNA-dependent interaction with wild-typeand mutant ATXN2. The immunoprecipitates also show co-IP of DDX6 andPABPC1, both ATXN2 interactors.

FIG. 2A is a representative micrograph of Staufen antibodyimmunostaining of cerebellum of wild-type mouse at 6 weeks of age.Expression of Staufen1 in cerebellum is observed (20× magnification).

FIG. 2B is a representative blot of silencing achieved for Staufen1.Staufen antibody demonstrates specificity to human and mouse endogenousStaufen1. Human HEK-293 cells or mouse N2a neuroblastoma cells weretransfected with control siRNAs or siRNAs directed against human ormouse Staufen1. For human siStaufen1, HEK-293 cells were harvested at 4days post-transfection. Protein extracts were analyzed by western blotto measure the silencing achieved for Staufen1. Blots were re-probed forβ-Actin as an internal loading control. The blot represents one of threeindependent experiments.

FIG. 2C is a representative blot of silencing achieved for Staufen1.Staufen1 antibody demonstrates specificity to human and mouse endogenousStaufen1. Human HEK-293 cells or mouse N2a neuroblastoma cells weretransfected with control siRNAs or siRNAs directed against human ormouse Staufen1. For mouse siStaufen1, N2a cells were harvested at 4 dayspost-transfection. Protein extracts were analyzed by western blot tomeasure the silencing achieved for Staufen1. Blots were re-probed forβ-Actin as an internal loading control. The blot represents one of threeindependent experiments.

FIG. 3A depicts a Western blot analysis of SCA2 patient-derived skinfibroblasts.

FIG. 3B depicts a Western blot analysis of lymphoblastoid B (LB) cellextracts.

FIG. 3C depicts a Western blot analysis of BAC-Q72 cerebellar extracts(24 weeks of age).

FIG. 3D depicts a Western blot analysis where overexpression of Flagtagged-ATXN3-Q56 in HEK-293 cells shows unaltered Staufen1 levelcompared with wild-type ATXN3.

FIG. 3E depicts a Western blot analysis showing increased abundance ofStaufen1 in SCA2 cells is unlinked to wild-type ATXN2 loss. HEK-293cells were transfected with siATXN2 RNA and analyzed by western blottingat 4 days post-transfection. Reduced ATXN2 levels do not result inalteration of Staufen1 steady-state levels compared with control siRNA.

FIG. 3F is a graph of the quantification of abundances on western blotsdetermined densitometrically.

FIG. 3G is a graph of qPCR analyses of cerebellar RNAs showing unalteredStaufen1 transcript levels in SCA2^(−/−) mouse cerebellum compared withSCA2^(+/+) mouse (8 weeks of age).

FIG. 4A depicts a Western blot analysis where cells were transfectedwith plasmids: Flag tagged-ATXN2 containing Q22 or Q58 or Q108 repeatsand protein extracts from harvested cells at 48 hr post-transfection tomeasure Staufen1 steady-state levels.

FIG. 4B depicts a blot showing stability of Staufen1 in SCA2fibroblasts. Normal and SCA2 fibroblasts were exposed to CHX (25 μg/ml)for 0 to 24 hr, and protein extracts were immunoblotted for Staufen1 foreach time point. Staufen1 levels are calculated as a percentage of the 0time point and p21 Waf1/Cip1 protein abundances were measured as anexperimental control to verify protein synthesis inhibition by CHX.Staufen1 protein stability is prolonged in SCA2 fibroblasts whencompared with normal fibroblasts.

FIG. 4C is a graph of the Quantification of Staufen1 on western blotsdetermined densitometrically.

FIG. 5A is a graph showing that SCA2-LB cells demonstrate decreased PCP2transcripts. qPCR analyses of synthesized cDNAs from SCA2-LB cells showsignificant reduction of PCP2 mRNA abundance when compared with normalLB cells. ATXN2 mRNA levels remain unchanged in both normal and SCA2-LBcells.

FIG. 5B is a blot showing that increased Staufen1 level in human cellculture independently induces reduction of PCP2 and CALB1 mRNAsabundances. HEK-293 cells were transfected with Flag-tagged Staufen1construct and harvested as two aliquots at 48 hr post-transfection.Protein extracts from one aliquot were immunoblotted to measure therelative expression of Flag-Staufen1.

FIG. 5C is a graph of qPCR analyses of synthesized cDNAs from secondaliquot showing decreased PCP2 and CALB1 transcripts compared withcontrol transfections. ATXN2 transcripts were not altered upon Staufen1overexpression. The data are means±SD, **p<0.01 (Student's t-test).

FIG. 5D illustrates a schematic of MYC tagged-PCP2 cDNA[(5′+3′)UTRs] (i)and PCP2 cDNA(+5′UTR) (ii).

FIG. 5E is a blot showing that increased Staufen1 levels inducesrepression of PCP2 protein synthesis. MYC tagged-PCP2 cDNA[(5′+3′)UTRs](5Di) PCP2 cDNA(+5′UTR) (5Dii) were cloned under CMV promoter andtransfected into short-term hygromycin selected HEK-293 cells expressingFlag-Staufen1. Forty-eight hrs post-transfection, western blot analysesshow significant reduction of exogenous PCP2 levels in Flag-Staufen1expressing cells transfected with MYC-tagged PCP2 cDNA[(5′+3′)UTRs](5Di) compared with control. Conversely, increased Staufen1 levels didnot show significant regulatory effect on expression of MYC-tagged PCP2cDNA(−3′UTR) (5Dii) when compared with control.

FIG. 5F is a graph of the quantification of MYC-tagged PCP2 on westernblots determined densitometrically. To control for equal transfection,Neomycin levels were measured which is expressed as an independentcassette in the MYC-tagged PCP2 plasmids. Blots were re-probed forβ-Actin as an internal loading control. The data are means±SD, **p<0.01(Student's t-test).

FIG. 6A is a blot where non-RNase treated HEK-293 whole cell extractsexpressing Flag or Flag-Staufen1 were subjected to immunoprecipitationwith Flag mAb beads. Bound protein-RNA complexes were eluted by Flagpeptide competition and IP products were divided equally into two partsand subjected to western blot and RT-PCR/qPCR analyses. Western blotanalyses of the eluted proteins shows co-IP of endogenous ATXN2 withFlag-Staufen1. RT-PCR analyses of the second aliquot shows thatFlag-Staufen1 pulls down PCP2 and CALB1 mRNAs but not GAPDH mRNA.

FIG. 6B depicts a schematic of DIG-labelled human PCP2 transcripts: PCP2[(5′+3′)UTR] (i), PCP2 (3′UTR) (ii) and PCP2 (5′UTR) (iii).

FIG. 6C is a blot of extracts from BL21<DE3> bacteria transformed withpET vectors containing His tagged-Staufen1, -GFP or pET that were run onSDS-PAGE and stained with coomassie brilliant blue or western blottedwith anti-His antibody (blot 1). For northwestern blot assay, bacterialprotein extracts: pET, His-GFP or His-Staufen1 were run on SDS-PAGE andtransferred onto PVDF membrane. The membranes were re-natured andhybridized with DIG-labelled PCP2 RNA probes as indicated. Followinganti-DIG antibody staining, the signals were detected by WesternChemiluminescent method. The results demonstrate Staufen1 binds directlyto PCP2 RNAs: (5′+3)UTRs (i) (blot 2) or (3′UTR) (ii) (blot 3) but notto PCP2(5′UTR) RNA (blot 4). His-GFP does not show any interactions withPCP2 RNA probes. The blot represents one of three independentexperiments.

FIG. 7A is a blot of Normal (ATXN2-Q22/22) and SCA2-LB (ATXN2-Q22/52)cells that were electroporated with siSTAU1 and harvested as twoaliquots for protein and RNA analyses after 4 days post-electroporation.Protein extracts from one aliquot were immunoblotted for Staufen1.

FIG. 7B is a graph of Western blot quantification determineddensitometrically of Staufen1/Actin in normal LB cells and SCA2-LBcells.

FIG. 7C is a graph of isolated RNAs from the second aliquot that weresubjected to qPCR analyses to measure PCP2 transcript levels. Staufen1depletion does prevent PCP2 transcript expression defects in SCA2-LBcells but not showing significant PCP2 transcript alterations in normalcells when compared with control siRNAs.

FIG. 7D is a graph showing silencing of Staufen1 does not showalteration of ATXN2 transcript steady-state levels in normal and SCA2-LBcells as judged by qPCR analyses.

FIG. 7E is a blot of SCA2-fibroblasts (ATXN2-Q22/42) cells that weretransfected or electroporated with siATXN2 at dose dependent fashion andharvested as two aliquots for protein and RNA analyses after 4 dayspost-transfection/electroporation. Protein extracts from one aliquotwere immunoblotted for ATXN2 and Staufen1 and showing significantreductions of Staufen1 abundance upon depletion of ATXN2 dosages whencompared with control siRNA.

FIG. 7F is a graph of the Quantification of western blots of FIG. 7Edetermined densitometrically.

FIG. 7G is a qPCR analysis of synthesized cDNAs from second aliquot showsignificant incremental of PCP2 transcript levels in SCA2-fibroblasts,upon depletion of ATXN2 dosages, when compared with control siRNA.

FIG. 7H is a blot of SCA2-LB (ATXN2-Q22/52) cells that were transfectedor electroporated with siATXN2 at dose dependent fashion and harvestedas two aliquots for protein and RNA analyses after 4 dayspost-transfection/electroporation. Protein extracts from one aliquotwere immunoblotted for ATXN2 and Staufen1 and showing significantreductions of Staufen1 abundance upon depletion of ATXN2 dosages whencompared with control siRNA.

FIG. 7I is a graph of the Quantification of western blots of FIG. 7Hdetermined densitometrically.

FIG. 7J is a qPCR analysis of synthesized cDNAs from second aliquot showsignificant incremental of PCP2 transcript levels in SCA2-LB cells, upondepletion of ATXN2 dosages, when compared with control siRNA.

FIG. 8A is a blot showing Staufen1 depletion restored autophagic pathwayproteins in ATXN2-Q22/58 knock-in cells. Cells were transfected withSTAU1 RNAi and analyzed by western blotting. Autophagy proteinsevaluated included the following: total (MTOR) and activated (pMTOR)mechanistic target of rapamycin which is an inhibitor of autophagy,SQSTM1/p62, or sequestosome-1, which binds proteins targeted to theautophagosome. LC3, a commonly used marker of autophagy function. LC3-Iis cytoplasmic while a cleaved form with higher mobility. LC3-II,localizes to the autophagosome. The amount of LC3-II is an indicator ofautophagosome quantity which typically increases when autophagy isdefective.

FIG. 8B is a blot showing antisense oligonucleotides (ASOs) targetingStaufen1 lower its expression in HEK-293-ATXN2[Q22/Q22] cells. STAU1ASO-A (5′-TCTCATGTTGTAGTTATAGG-3′ used at the indicated dose reducedSTAU1 levels, determined by western blotting. Expression relative toactin.

FIG. 8C is a blot showing antisense oligonucleotides (ASOs) targetingStaufen1 lower its expression in HEK-293-ATXN2[Q22/Q22] cells. STAU1ASO-B (5′-CTGGAAAGATAGTCCAGTTG-3′) used at the indicated dose reducedSTAU1 levels, determined by western blotting. Expression relative toactin.

FIG. 9A is a histogram representing quantities and sizes of co-localizedgranules.

FIG. 9B presents images of co-localization of Staufen1 with mutant ATXN2detected in ATXN2^(Q127) mouse cerebellar PCs (24 wks of age). Combineddouble-staining of Staufen1 (red) and ATXN2 (green) shows the presenceof aggregates (white arrows) in ATXN2^(Q127) mice.

FIG. 10A presents a western blot analysis of SCA2 fibroblasts showingincreased Staufen1 levels compared with normal controls. DDX6 levels areunchanged.

FIG. 10B presents a western blot analysis of LBCs showing increasedStaufen1 levels compared with normal controls. DDX6 levels areunchanged.

FIG. 10C presents a western blot analysis showing increased Staufen1levels in cerebellar extracts from ATXN2^(Q127) mice at 24 weeks of age.

FIG. 10D presents a western blot analysis showing increased Staufen1levels in cerebellar extracts from BAC-Q72 mice at 24 weeks of age.

FIG. 10E presents a western blot analysis of cerebellar extracts fromTDP-43^(Tg/+) hemizygous mice at 8 weeks of age compared to wild-typelitter mates.

FIG. 10F presents a western blot analysis of spinal cord extracts fromTDP-43^(Tg/+) hemizygous mice at 8 weeks of age compared to wild-typelitter mates.

FIG. 10G presents a western blot analysis showing increased Staufen1 inFB extracts from a patient with ALS caused by TDP-43^(G298S) mutation.

FIG. 10H presents a western blot analysis showing arsenite-inducedstress results in exaggerated increases in STAU1 levels in HD and SCA3FBs. SCA2, HD and SCA3 FBs were treated with sodium arsenite (1.0 mM)for 0, 3 and 6 hr followed by immunoblotting. β-actin was used asloading control and representative blots of three independentexperiments are shown.

FIG. 11A is a graph showing Staufen1 transcript levels do not correspondto the differences in the steady state Staufen1 protein levels observedin SCA2-FBs when compared to control cells.

FIG. 11B is a graph showing Staufen1 transcript levels do not correspondto the differences in the steady state Staufen1 protein levels observedin SCA2-LBCs when compared to control cells.

FIG. 11C is a graph showing a qRT-PCR analysis of cerebellar RNAs fromATXN2^(Q127) mice showing unaltered Staufen1 transcript levels comparedto wild-type littermates at 24 weeks of age. Gene expression levels werenormalized to Actb.

FIG. 11D is a graph showing a qRT-PCR analysis of cerebellar RNAs fromBAC-Q72 mice showing unaltered Staufen1 transcript levels compared towild-type littermates at 24 weeks of age. Gene expression levels werenormalized to Actb.

FIG. 12 is a western blot analysis where normal and SCA2 FBs wereexposed to CHX (25 μg/ml) for different times from 0 to 24 hr, andprotein extracts were immunoblotted for Staufen1. Staufen1 levels arequantified as a percentage of the value for the 0 time point and p21Waf1/Cip1 protein levels are shown to verify CHX inhibition. Staufen1protein stability was prolonged in SCA2 FBs compared to normal FBs.Loading control, β-actin found to be invariant over the treatmentperiod. Representative blots of three independent experiments are shown.

FIG. 13A is a western blot analysis illustrating that Autophagy isimpaired in SCA2 FBs as indicated by increased levels of processedLC3-II.

FIG. 13B is a plot illustrating Flag immunoprecipitation of HEK-293 cellextracts expressing Flag-tagged STAU1 (non-RNAse A treated).Flag-Staufen1 pulled down ATXN2 protein, and PCP2 and mTOR mRNAs onwestern blot and RT-PCR.

FIG. 13C is a western blot analysis illustrating Introduction of anexpanded ATXN2 allele into HEK-293 cells by CRISPR/Cas9 editing resultsin STAU1 increase and autophagic dysfunction as shown by increases inmTOR, P-mTOR, p62 and processed LC3-II on western blots. Thedifferentially regulated cerebellar transcript PCP2 is decreased in thiscell model. β-actin was used as loading control.

FIG. 14A is a schematic where single guide RNA (sgRNA) sequence (green),the PAM sequence (NGG) (red) and Cas9 cleavage site (red arrowhead) areshown. Cas9 cleaves the DNA at the target site with the help of sgRNAguidance. The left and right arms of the target donor insert CAG58repeats into the ATXN2 locus through HDR. RT-PCR screening forATXN2-CAG22/58 [ATXN2-Q22/58] Knockin-positive clones using theindicated primers. HEK-293 cells were co-transfected with linearizeddonor vector and single guide RNA pgRNA-Cas9 vector. Puromycin selectedcells were titrated in 96-well plates at 1 cell/well, and then expandedand maintained for PCR screening to identify knock-in positive cells.

FIG. 14B presents RT-PCR analyses of some identified ATXN2-Q22/58Knock-in clones.

FIG. 14C is a graph showing CRISPR/Cas9 edited ATXN2-Q22/58 KI cellsmirror SCA2 phenotypes including Staufen1 abundance (FIG. 13B; westernblotting) and dysregulation of transcripts (PCP2, CACNA1G and ITPR1)associated with SCA2 in vivo analyzed by qRT-PCR. Data are means±SD,***P<0.01, Student t-test.

FIG. 14D presents images showing ATXN2 and Staufen1 co-localize inSG-like aggregates. Representative immunohistochemical images fromwild-type and ATXN2-Q22/58 KI cells stained with antibodies directedagainst Staufen1 (green) and ATXN2 (red) showing presence of SG-likestructures positive for both ATXN2 and Staufen1. All images for arespective antibody were taken at the same exposure times. Scale bar=100μM.

FIG. 15A is a western blot analysis of Mutant ATXN2 overexpressedHEK-293 cells showing increased levels of Staufen1, P-mTOR, p62 andprocessed LC3-II.

FIG. 15B is a western blot analysis showing Bafilomycin A1 lowersStaufen1 clearance. HEK-293 and ATXN2-Q22/58 knock-in cells were treateddose-wise with Baf for 6 h and analyzed by western blotting. Baftreatment showing dose-wise increased levels of processed LC3-II alongwith p62 and Staufen1 for both cell types compared to untreated cells.

FIG. 15C is a western blot analysis of Staufen1 overexpressed HEK-293cell extracts showing reduced PCP2 protein levels (differentiallyregulated cerebellar transcript Pcp2) and increased P-mTOR, mTOR, p62and LC3-II levels. β-actin was used as loading control andrepresentative blots of three independent experiments are shown.

FIG. 16A is a western blot analysis showing Staufen1 depletion restoresautophagic pathway proteins in ATXN2-Q22/58 KI cells. Cells weretransfected with STAU1 siRNA and analyzed by western blotting. Staufen1depleted ATXN2-Q22/58 KI cells show restoration of autophagic pathwayproteins without affecting ATXN2 steady-state levels.

FIG. 16B is a western blot analysis showing ATXN2 siRNA lowers Staufen1abundance, and normalizes autophagic pathway proteins in ATXN2-Q22/58 KIcells.

FIG. 16C is a graph showing Stau1 haploinsufficiency improves abnormalmotor behavior of ATXN2^(Q127) mice as determined by rotarod behavior at8, 12, 16 and 20 weeks of age. ATXN2^(Q127); Stau1^(+/−) mice (green)have improved rotarod performances compared with ATXN2^(Q127) mice (red)starting at 12 through 20 weeks of age; N=9-15 mice per group. Valuesshown are mean±SE. Significance was determined using generalizedestimating equations (GEE). NS, nonsignificant, *P<0.05, **P<0.01.

FIG. 16D is a western blot analysis of cerebellar extracts fromwild-type and transgenic animals with normal and 50% Staufen1 reductionat 20 weeks of age. The PC proteins tightly correlate with diseaseprogression in two SCA2 mouse models. Phosphorylated mTor, total mTor,p62, and LC3-II are markers of autophagosome function. Each lanerepresents a cerebellar extract from an individual mouse. β-actin isused as a loading control and the blots are from three replicateexperiments.

FIG. 16E is a graph of a quantitative analysis of western blots shown inFIGS. 16D and 20. Data are means±SD, ***P<0.001, Student t-test.

FIG. 16F is a graph of a quantitative analysis of western blots shown inFIGS. 16D and 20. Data are means±SD, ***P<0.001, Student t-test.

FIG. 17A is western blot analysis where HEK-293 and ATXN2-Q22/58 KIcells were treated dose-wise with Rapamycin for 24 h followed by westernblotting. Rap-induced mTOR inhibition dose-wise decreased levels ofelevated Staufen1 along with p62 and LC3-II in ATXN2-Q22/58 KI cellswithout affecting those levels in control HEK-293 cells. β-actin wasused as loading control and representative blots of three independentexperiments are shown.

FIG. 17B is a western blot analysis showing RNAi-mediated mTOR depletionlowers Staufen1 and improves autophagy marker proteins. HEK-293 andATXN2-Q22/58 KI cells were transfected with mTOR RNAi and cells wereanalyzed by western blotting at 4 days post-transfection. mTORRNAi-mediated mTOR and phospho-mTOR depletion showing reduced Staufen1and autophagic pathway protein component levels in ATXN2-Q22/58 KI cellswithout affecting those levels in control HEK-293 cells. β-actin wasused as loading control and representative blots of three independentexperiments are shown.

FIG. 18A is a western blot analysis where Normal (ATXN2-Q22/22) and SCA2(ATXN2-Q22/52) LBCs were electroporated with STAU1 RNAi and Staufen1levels were determined by western blotting after 4 dayspost-electroporation.

FIG. 18B is a graph of the quantification of the blot in FIG. 18A.

FIG. 18C is a graph illustrating Staufen1 depletion was associated withincreased PCP2 transcript levels in SCA2-LBCs and where matched cultureswere used for qRT-PCR analyses to measure PCP2 transcript levels. PCP2mRNA was unresponsive to Staufen1 knockdown in normal LBCs. Staufen1silencing did not alter ATXN2 transcript steady-state levels in normaland SCA2-LBCs. β-actin and GAPDH RNA were used as internal controls forwestern blot and qRTPCR analyses, respectively. Abundances relative toactin determined densitometrically. The results from three independentexperiments are shown. Data are means±SD, **P<0.01, Student t-test.

FIG. 19A is a graph illustrating Staufen1 overexpression reduced PCP2mRNA abundance. Total RNA was isolated from the one aliquot of harvestedHEK-293 cells (FIG. 15C) overexpressing Flag-tagged Staufen1 andsubjected to qRT-PCR analyses. Cells overexpressing Staufen1 havedecreased mRNAs for PCP2 but not ATXN2 compared with controls. Geneexpression levels were normalized to GAPDH.

FIG. 19B is a graph of the co-transfection of Flagtagged STAU1 withluciferase-PCP2-3′UTR and Renilla luciferase expression plasmids inSHSY5Y cells demonstrating reduction of luciferase activity and wherethe 3′UTR of PCP2 was cloned downstream of luciferase expressionplasmid. Luciferase expression was normalized with Renilla luciferaseexpression to control transfection equalities. Data are means±SD,**P<0.01, ***P<0.001, Student t-test.

FIG. 20 is a Western blotting analysis of cerebellar extracts fromcrossed ATXN2Q127; Stau1−/− at 18 weeks of age showing restoration ofCalb1, Pcp2, Pcp4, Rgs8, Homer3, Fam107b and autophagic pathwayproteins. Each lane represents cerebellar extract from an individualmouse. β-actin was used as loading control and the blots are from threereplicate blots. Quantitative analysis of western blots shown in FIGS.16E-16F.

FIG. 21A is a Western blotting analysis showing that STAU1 is elevatedin a patient fibroblast line with TDP-43 G298S mutation (loaded twice).SCA2 FBs serve as a positive control.

FIG. 21B is a Western blotting analysis showing that STAU1 is elevatedin HEK293 cells overexpressing nonmutant TDP-43 or truncated TDP-25.

FIG. 21C is a Western blotting analysis showing that STAU1 is elevatedin HEK293 cells overexpressing mutant forms of TDP-43.

FIG. 21D is a Western blotting analysis showing that STAU1 and LC3-IIare increased in HEK293 transfected with non-mutant or mutant TDP-43.

FIG. 22A is a Western blotting analysis showing that STAU1 is elevatedin three different patient fibroblasts with expanded C9ORF72 (1 PD, 2&3FTD) vs 3 different control lines. STAU1 increase is also seen in a PDpatient fibroblast with a Tau (MAPT) mutation.

FIG. 22B is a Western blotting analysis showing that STAU1 and LC3-IIare elevated in HEK293 cells overexpressing normal repeat C9ORF72.TDP-43 was included as a positive control.

FIG. 22C is a Western blotting analysis showing that overexpression ofTau (P301L) or APP (Swe/Ind mutations) in HEK293 cells increased STAU1levels. Upper blot detected with GFP, FLAG, & Actin antibodies. TDP-43was included as a positive control.

FIG. 23A depicts a western blot analysis of extracts from ALS FBs withTDP-43 show increased STAU1 levels compared with normal controls. All FBextracts show increased mTOR and P-mTOR levels and increased p62 andLC3-II levels compared with control FBs. β-Actin was used as loadingcontrol and representative blots of three independent experiments areshown.

FIG. 23B depicts a western blot analysis of extracts from ALS FBs withC9orf72 mutations show increased STAU1 levels compared with normalcontrols. All FB extracts show increased mTOR and P-mTOR levels andincreased p62 and LC3-II levels compared with control FBs. β-Actin wasused as loading control and representative blots of three independentexperiments are shown.

FIG. 23C depicts a western blot analysis of extracts from HD FBs showincreased STAU1 levels compared with normal controls. All FB extractsshow increased mTOR and P-mTOR levels and increased p62 and LC3-IIlevels compared with control FBs. β-Actin was used as loading controland representative blots of three independent experiments are shown.

FIG. 23D depicts a western blot analysis of extracts from AD FBs [(PSEN1mutations: M146I, E184D and Intron4 Var) and MAPT mutation: (N279K)]show increased STAU1 levels compared with normal controls. All FBextracts show increased mTOR and P-mTOR levels and increased p62 andLC3-II levels compared with control FBs. β-Actin was used as loadingcontrol and representative blots of three independent experiments areshown.

FIG. 23E depicts a western blot analysis of extracts from SCA2 FBs showincreased STAU1 levels compared with normal controls. All FB extractsshow increased mTOR and P-mTOR levels and increased p62 and LC3-IIlevels compared with control FBs. β-Actin was used as loading controland representative blots of three independent experiments are shown.

FIG. 24A depicts qRT-PCR analyses of ALS, AD, HD and SCA2 FBs revealedunaltered mRNA levels for STAU1 compared with control cells. Geneexpression levels were normalized to Actin.

FIG. 24B depicts qRT-PCR analyses of ALS, AD, HD and SCA2 FBs revealedunaltered mRNA levels for mTOR compared with control cells. Geneexpression levels were normalized to Actin.

FIG. 24C depicts qRT-PCR analyses of Stau1 and mTor mRNAs fromcerebellar (CB) and spinal cord (SC) RNAs from ATXN2^(Q127). (24 weeksof age; 3 animals per group) mice compared to wild-type littermates.

FIG. 24D depicts qRT-PCR analyses of Stau1 and mTor mRNAs fromcerebellar (CB) and spinal cord (SC) RNAs from TDP-43^(Tg/+) hemizygousmice (8 weeks of age; 3 animals per group) mice compared to wild-typelittermates.

FIG. 25A depicts a western blotting analysis of CRISPR/CAS9 editedATXN2-Q58 KI cells showing increased STAU1, mTOR levels and mTORactivity (increased P-mTOR, P-S6 and P-4E-BP1 levels) on western blot.

FIG. 25B depicts a western blotting analysis of HEK-293 cellstransfected with plasmid construct for 48 hrs expressing exogenouswild-type TDP-43. β-Actin was used as loading control and representativeblots of three independent experiments are shown.

FIG. 25C depicts a western blotting analysis of HEK-293 cellstransfected with plasmid construct for 48 hrs expressing exogenousmutant TDP-43 (G298S, A382T, G348C and CTF). β-Actin was used as loadingcontrol and representative blots of three independent experiments areshown.

FIG. 25D depicts a western blotting analysis of HEK-293 cellstransfected with plasmid construct for 48 hrs expressing exogenous TAU.β-Actin was used as loading control and representative blots of threeindependent experiments are shown.

FIG. 26A depicts a western blotting analysis of ATXN2^(Q127) mousecerebellar extracts (24 weeks of age; 3 animals per group) showingincreased Stau1, mTor and P-mTor, p62 and LC3-II levels compared withwild-type litter mate controls. Each lane represents an individualmouse. β-Actin was used as a loading control and the blots are fromthree replicate experiments.

FIG. 26B depicts a western blotting analysis of ATXN2^(Q127) mousecerebellar extracts from TDP-43^(Tg/+) hemizygous mice (8 weeks of age;3 animals per group) showing increased Stau1, mTor and P-mTor, p62 andLC3-II levels compared with wild-type litter mate controls. Each lanerepresents an individual mouse. β-Actin was used as a loading controland the blots are from three replicate experiments.

FIG. 26C depicts a western blotting analysis of ATXN2^(Q127) mousespinal cord extracts from TDP-43^(Tg/+) hemizygous mice (8 weeks of age;3 animals per group) showing increased Stau1, mTor and P-mTor, p62 andLC3-II levels compared with wild-type litter mate controls. Each lanerepresents an individual mouse. β-Actin was used as a loading controland the blots are from three replicate experiments.

FIG. 26D depicts a western blotting analysis of ATXN2^(Q127) mousecerebellar extracts from BAC-C9orf72 mouse brain extracts (20 weeks ofage; 5 animals per group) showing increased Stau1, mTor and P-mTor, p62and LC3-II levels compared with wild-type controls. Each lane representsan individual mouse. β-Actin was used as a loading control and the blotsare from three replicate experiments.

FIG. 27A depicts a western blotting analysis of HEK-293 cellsexogenously expressing Flag-tagged STAU1, which were analyzed 48 hrspost-transfection and show increased mTOR, P-mTOR, p62 and LC3-IIlevels.

FIG. 27B depicts a qRT-PCR analysis showing unchanged mTOR mRNA levels48 hrs post-transfection. RNA expression levels were normalized to ACTB.

FIG. 27C depicts an analysis of non-RNase A treated HEK-293 whole cellextracts expressing Flag or Flag-STAU1, which were immunoprecipitatedwith Flag mAb beads. Flag-STAU1 pulled down mTOR RNA by RT-PCR analysis.

FIG. 27D is a schematic of DIG-labeled human mTOR RNA probes with 5′UTRand 3′UTR sequences.

FIG. 27E is a blot illustrating that STAU1 directly interacts withmTOR-5′UTR RNA (blot 2), but not with mTOR-3′UTR RNA (blot 3).Bacterially expressed His-STAU1, His-GFP, or control bacterial lysatewere stained with Coomassie brilliant blue (CBB) on SDS-PAGE or westernblotted with anti-His antibody (blot 1). Protein blots were hybridizedwith DIG-labelled mTOR RNA probes followed by anti-DIG antibodystaining. His-GFP shows no interactions.

FIG. 27F is a schematic of mTOR-5′UTR-LUC and PCP2-5′UTR-LUC (control)reporters.

FIG. 27G presents a western blotting analysis showing exogenousexpression of STAU1.

FIG. 27H presents data illustrating that exogenous STAU1 expressioninduces increased expression of the mTOR-5′UTR-LUC construct but notPCP2-5′UTR-LUC (PCP2-3′UTR substrate for SMD) on luciferase reporterassay.

FIG. 27I is a schematic representation of STAU1 and mutant STAU1excluding RBD3 (STAU1[RBDΔ3]).

FIG. 27J presents a western blotting analysis of HEK-293 cells that weretransfected with STAU1 constructs alone or co-transfected withmTOR-5′UTR-LUC reporter construct followed by western blotting.Significantly increased endogenous mTOR level was achieved only throughthe STAU1 construct retaining the RBD3.

FIG. 27K presents data of HEK-293 cells that were transfected with STAU1constructs alone or co-transfected with mTOR-5′UTR-LUC reporterconstruct followed by luciferase assay. Significantly increasedluciferases activity was achieved only through the STAU1 constructretaining the RBD3.

FIG. 28A presents a western blotting analysis of HEK-293 cells that weretreated with thapsigargin and analyzed by western blotting. Thetreatment increased STAU1, mTOR and P-mTOR levels, and reduced autophagyactivity (increased p62 and LC3-II levels). β-Actin was used as loadingcontrol. Representative blots of three independent experiments are shown

FIG. 28B presents a western blotting analysis of HEK-293 cells that weretreated with tunicamycin and analyzed by western blotting. The treatmentincreased STAU1, mTOR and P-mTOR levels, and reduced autophagy activity(increased p62 and LC3-II levels). β-Actin was used as loading control.Representative blots of three independent experiments are shown

FIG. 28C presents a western blotting analysis of HEK-293 cells that weretreated with ionomycin and analyzed by western blotting. The treatmentincreased STAU1, mTOR and P-mTOR levels, and reduced autophagy activity(increased p62 and LC3-II levels). β-Actin was used as loading control.Representative blots of three independent experiments are shown

FIG. 28D presents a western blotting analysis of HEK-293 cells that weretreated with sodium arsenite and analyzed by western blotting. Thetreatment increased STAU1, mTOR and P-mTOR levels, and reduced autophagyactivity (increased p62 and LC3-II levels). β-Actin was used as loadingcontrol. Representative blots of three independent experiments are shown

FIG. 28E presents a western blotting analysis of HEK-293 cells that weretreated with hyperthermia and analyzed by western blotting. Thetreatment increased STAU1, mTOR and P-mTOR levels, and reduced autophagyactivity (increased p62 and LC3-II levels). β-Actin was used as loadingcontrol. Representative blots of three independent experiments areshown.

FIG. 28F presents data of mouse cortical neurons treated withthapsigargin that also showed increased Stau1, mTor, P-mTor, p62, andLC3-II (left), but neurons null for Stau1 are resistant tothapsigargin-mediated induction of these genes (right). β-Actin was usedas loading control. Representative blots of three independentexperiments are shown.

FIG. 29A presents data illustrating that STAU1 depletion lowers mTORactivity concurrently with mTOR targets and restores autophagic pathwayproteins in ATXN2-Q22/58 KI cells. Cells were transfected with STAU1siRNA and analyzed by western blotting. STAU1 depletion in ATXN2-Q22/58KI cells results in decreased mTOR levels, mTOR activity (reducedP-mTOR, P-S6 and P-4E-BP1 levels), as well as p62 and LC3-II levels,indicating restored autophagy activity. β-Actin is used as a loadingcontrol and the blots are from three replicate experiments

FIG. 29B presents western blots of HEK-293 cells expressing Flag-taggedwild-type or mutant TDP-43 or empty vectors followed by transfectionwith STAU1 siRNA. STAU1 depletion lowers mTOR levels and restoresautophagic pathway proteins in cells with TDP-43. β-Actin is used as aloading control and the blots are from three replicate experiments.

FIG. 29C presents data of ALS-C9orf72 FBs transfected with STAU1 siRNAand analyzed by western blotting. STAU1 depletion lowers mTOR levels andrestores autophagic pathway proteins in cells with C9orf72 mutation.β-Actin is used as a loading control and the blots are from threereplicate experiments.

FIG. 30 presents data illustrating STAU1 depleted cells are resistant tomTOR activation by TDP-43. HEK-293 cells were treated 72 hrs by STAU1siRNA and then transfected with Flag-tagged wild-type or mutant TDP-43for an additional 48 hrs followed by western blotting. STAU1-depletedcells have resistance to mTOR activation upon overexpression ofdisease-linked genes compared with controls. 3-Actin is used as aloading control and the blots are from three replicate experiments.

FIG. 31A presents data illustrating that a reduction of Stau1 reducesmTor activity and restores autophagic pathway proteins in ATXN2^(Q127)mice. Western blotting of cerebellar extracts from a cross ofATXN2^(Q127) and Stau1^(+/−) mice (34 weeks of age) showing reduced mToractivity and normalization of autophagic pathway proteins in vivo. Eachlane represents extract from an individual mouse. β-Actin is used as aloading control and the blots are from three replicate experiments.

FIG. 31B presents data illustrating that a reduction of Stau1 reducesmTor activity and restores autophagic pathway proteins in ATXN2^(Q127)mice. Western blotting of cerebellar extracts from ATXN2^(Q127);Stau1^(+/−) mice (20 weeks of age) showing reduced mTor activity andnormalization of autophagic pathway proteins in vivo. Each lanerepresents extract from an individual mouse. β-Actin is used as aloading control and the blots are from three replicate experiments.

FIG. 31C presents western blotting of spinal cord extracts fromTDP-43^(Tg/+) hemizygous mice haploinsufficient for Stau1 (22 weeks ofage) showing reduced mTor activity and normalization of autophagicpathway proteins. Each lane represents extract from an individual mouse.β-Actin is used as a loading control and the blots are from threereplicate experiments.

FIG. 31D presents a quantitative analysis of western blots shown inFIGS. 31A and 31B. Data are means±SD, **P<0.01, ***P<0.001, Studentt-test.

FIG. 31E presents a quantitative analysis of western blots shown in FIG.31C. Data are means±SD, **P<0.01, ***P<0.001, Student t-test.

FIG. 32A is a graph of data for mouse NIH3T3 cells that were transfectedwith 250 nM of the indicated ASOs or phosphate buffered saline as anegative control and Stau1 expression was determined by quantitative PCRusing primers targeting exon 4, after 4 days following the transfection.All staufen ASOs target both human and mouse staufen1 sequences and hadthe 5-10-5 MOE gapmer chemistry.

FIG. 32B is a graph of data for Mouse NIH3T3 cells that were transfectedwith 250 nM of the indicated ASOs or phosphate buffered saline or siRNAand after 4 days Stau1 expression was determined by western blotting.All staufen ASOs target both human and mouse staufen1 sequences and hadthe 5-10-5 2′-O-methoxyethyl (MOE) gapmer chemistry.

FIG. 32C is a graph of data for SCA2 patient derived skin cellfibroblasts (SCA2-03[Q22/Q35]) that were transfected with the indicatedconcentrations of the indicated ASOs or phosphate buffered saline andafter 4 days STAU1 expression was determined by quantitative PCR. Allstaufen ASOs target both human and mouse staufen1 sequences and had the5-10-5 2′-MOE gapmer chemistry.

FIG. 32D presents data of TDP-43+/− transgenic mice that were treated byintracerebroventricular injection with 250 mg of the indicated ASOs orsaline. After 15 days treatment the spinal cords were removed and Stau1expression was determined by quantitative PCR. All staufen ASOs targetboth human and mouse staufen1 sequences and had the 5-10-5 2′-MOE gapmerchemistry. Values are means and standard deviations. N number mice were5 (PBS), 4 (UU000181), 1 (UU00186), 2 (UU00045), 4 (UU00193), 5(UU00199).

FIG. 32E presents data of microgliosis that was assessed by determiningAif1 expression, revealing no increased Aif1 expression compared to thecontrol, or no evidence for microgliosis. All staufen ASOs target bothhuman and mouse staufen1 sequences and had the 5-10-5 2′-MOE gapmerchemistry. Values are means and standard deviations. N number mice were5 (PBS), 4 (UU000181), 1 (UU00186), 2 (UU00045), 4 (UU00193), 5(UU00199).

DESCRIPTION OF EMBODIMENTS

Although the following detailed description contains many specifics forthe purpose of illustration, a person of ordinary skill in the art willappreciate that many variations and alterations to the following detailscan be made and are considered to be included herein. Accordingly, thefollowing embodiments are set forth without any loss of generality to,and without imposing limitations upon, any claims set forth. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs.

As used in this written description, the singular forms “a,” “an” and“the” include express support for plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a polymer”can include a plurality of such polymers.

As used herein, “subject” refers to a mammal that can benefit fromtreatment with a Staufen1-regulating agent. A benefit can be obtained ifthe subject has a disease or condition, or is at risk of developing adisease or condition for which a Staufen1-regulating agent is atherapeutically effective treatment or preventative measure. In someaspects, such subject may be a human.

As used herein, the terms “treat,” “treatment,” or “treating” when usedin conjunction with the administration of a Staufen1-regulating agent,such as an siRNA or anti-sense oligonucleotide (ASO) that targets theSTAU1 gene, including compositions and dosage forms thereof, refers toadministration to subjects who are either asymptomatic or symptomatic.In other words, “treat,” “treatment,” or “treating” can be to reduce,ameliorate or eliminate symptoms associated with a condition present ina subject, or can be prophylactic, (i.e. to prevent or reduce theoccurrence of the symptoms in a subject). Such prophylactic treatmentcan also be referred to as prevention of the condition. Treatmentoutcomes can be expected or unexpected. In one specific aspect, atreatment outcome can be a delay in occurrence or onset of a disease orconditions or the signs or symptoms thereof. In another aspect, atreatment can be reducing, ameliorating, eliminating, or otherwiseproviding a subject with relief from (i.e. relieving) the condition withwhich they are afflicted, or providing relief from signs or symptoms ofthe condition.

As used herein a “therapeutic agent” refers to an agent that can have abeneficial or positive effect on a subject when administered to thesubject in an appropriate or effective amount.

As used herein, the terms “inhibit” or “inhibiting” are used to refer toa variety of inhibition techniques. For example, the terms “inhibit” or“inhibiting” can refer to pre- and/or post-transcriptional inhibition.With respect to pre-transcription inhibition, “inhibit” or “inhibiting”can refer to preventing or reducing transcription of a gene, inducingaltered transcription of a gene, and/or reducing a rate of transcriptionof a gene, whether permanent, semi-permanent, or transient. Thus, insome examples, “inhibit” or “inhibiting” can refer to permanent changesto the DNA, whereas in other examples no permanent change to the DNA ismade. With respect to post-transcriptional inhibition, “inhibit” or“inhibiting” can refer to preventing or reducing translation of agenetic sequence to a protein, inducing an altered translation of agenetic sequence to an altered protein (e.g. as misfolded protein,etc.), and/or reducing a rate of translation of a genetic sequence to aprotein, whether permanent, semi-permanent, or transient. In somespecific examples, “inhibit” or “inhibiting” can refer topre-transcriptional inhibition. In other specific examples, “inhibit” or“inhibiting” can refer to post-transcriptional inhibition. Of course,the type of inhibition can depend on the specific type(s) ofinhibitor(s) or therapeutic agent(s) employed. Thus, “inhibit” or“inhibiting” can include any decrease in expression of a gene ascompared to native expression, whether pre- or post-transcriptional,partial or complete.

As used herein, the terms “formulation” and “composition” are usedinterchangeably and refer to a mixture of two or more compounds,elements, or molecules. In some aspects the terms “formulation” and“composition” may be used to refer to a mixture of one or more activeagents with a carrier or other excipients. Compositions can take nearlyany physical state, including solid, liquid (i.e. solution), or gas.Furthermore, the term “dosage form” can include one or moreformulation(s) or composition(s) provided in a format for administrationto a subject.

The phrase “effective amount,” “therapeutically effective amount,” or“therapeutically effective rate(s)” of an active ingredient refers to anon-toxic, but sufficient amount or delivery rates of the activeingredient or therapeutic agent, to achieve therapeutic results intreating a disease or condition for which the drug is being delivered.It is understood that various biological factors may affect the abilityof a substance to perform its intended task. Therefore, an “effectiveamount,” “therapeutically effective amount,” or “therapeuticallyeffective rate(s)” may be dependent in some instances on such biologicalfactors. Further, while the achievement of therapeutic effects may bemeasured by a physician or other qualified medical personnel usingevaluations known in the art, it is recognized that individual variationand response to treatments may make the achievement of therapeuticeffects a subjective decision. The determination of a therapeuticallyeffective amount or delivery rate is well within the ordinary skill inthe art of pharmaceutical sciences and medicine. See, for example,Meiner and Tonascia, “Clinical Trials: Design, Conduct, and Analysis,”Monographs in Epidemiology and Biostatistics, Vol. 8 (1986).

As used herein, a “dosing regimen” or “regimen” such as “treatmentdosing regimen,” or a “prophylactic dosing regimen,” refers to how,when, how much, and for how long a dose of a composition can or shouldbe administered to a subject in order to achieve an intended treatmentor effect.

As used herein, “carrier,” and “pharmaceutically acceptable carrier” maybe used interchangeably, and refer to any liquid, gel, salve, solvent,liquid, diluent, fluid ointment base, liposome, micelle, giant micelle,or the like, or any other suitable carrier that is suitable for deliveryof a therapeutic agent to and/or into a target cell (e.g. a nerve cell)and for use in contact with a subject or the subject's tissue withoutcausing adverse physiological responses, and which does not interactwith the other components of the composition in a deleterious manner.

In this application, “comprises,” “comprising,” “containing” and“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like, and aregenerally interpreted to be open ended terms. The terms “consisting of”or “consists of” are closed terms, and include only the components,structures, steps, or the like specifically listed in conjunction withsuch terms, as well as that which is in accordance with U.S. Patent law.“Consisting essentially of” or “consists essentially of” have themeaning generally ascribed to them by U.S. Patent law. In particular,such terms are generally closed terms, with the exception of allowinginclusion of additional items, materials, components, steps, orelements, that do not materially affect the basic and novelcharacteristics or function of the item(s) used in connection therewith.For example, trace elements present in a composition, but not affectingthe compositions nature or characteristics would be permissible ifpresent under the “consisting essentially of” language, even though notexpressly recited in a list of items following such terminology. Whenusing an open ended term, like “comprising” or “including,” in thiswritten description it is understood that direct support should beafforded also to “consisting essentially of” language as well as“consisting of” language as if stated explicitly and vice versa.

The terms “first,” “second,” “third,” “fourth,” and the like in thedescription and in the claims, if any, are used for distinguishingbetween similar elements and not necessarily for describing a particularsequential or chronological order. It is to be understood that any termsso used are interchangeable under appropriate circumstances such thatthe embodiments described herein are, for example, capable of operationin sequences other than those illustrated or otherwise described herein.Similarly, if a method is described herein as comprising a series ofsteps, the order of such steps as presented herein is not necessarilythe only order in which such steps may be performed, and certain of thestated steps may possibly be omitted and/or certain other steps notdescribed herein may possibly be added to the method.

As used herein, the term “substantially” refers to the complete ornearly complete extent or degree of an action, characteristic, property,state, structure, item, or result. For example, an object that is“substantially” enclosed would mean that the object is either completelyenclosed or nearly completely enclosed. The exact allowable degree ofdeviation from absolute completeness may in some cases depend on thespecific context. However, generally speaking the nearness of completionwill be so as to have the same overall result as if absolute and totalcompletion were obtained. The use of “substantially” is equallyapplicable when used in a negative connotation to refer to the completeor near complete lack of an action, characteristic, property, state,structure, item, or result. For example, a composition that is“substantially free of” particles would either completely lackparticles, or so nearly completely lack particles that the effect wouldbe the same as if it completely lacked particles. In other words, acomposition that is “substantially free of” an ingredient or element maystill actually contain such item as long as there is no measurableeffect thereof.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint. Unless otherwise stated,use of the term “about” in accordance with a specific number ornumerical range should also be understood to provide support for suchnumerical terms or range without the term “about”. For example, for thesake of convenience and brevity, a numerical range of “about 50milligrams to about 80 milligrams” should also be understood to providesupport for the range of “50 milligrams to 80 milligrams.” Furthermore,it is to be understood that in this written description support foractual numerical values is provided even when the term “about” is usedtherewith. For example, the recitation of “about” 30 should be construedas not only providing support for values a little above and a littlebelow 30, but also for the actual numerical value of 30 as well.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the same list solely based ontheir presentation in a common group without indications to thecontrary.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 1 to about 5” should beinterpreted to include not only the explicitly recited values of about 1to about 5, but also include individual values and sub-ranges within theindicated range. Thus, included in this numerical range are individualvalues such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4,and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually.

This same principle applies to ranges reciting only one numerical valueas a minimum or a maximum. Furthermore, such an interpretation shouldapply regardless of the breadth of the range or the characteristicsbeing described.

Reference in this application may be made to compositions, systems, ormethods that provide “improved” or “enhanced” performance. It is to beunderstood that unless otherwise stated, such “improvement” or“enhancement” is a measure of a benefit obtained based on a comparisonto compositions, systems or methods in the prior art. Furthermore, it isto be understood that the degree of improved or enhanced performance mayvary between disclosed embodiments and that no equality or consistencyin the amount, degree, or realization of improvement or enhancement isto be assumed as universally applicable.

Reference throughout this specification to “an example” means that aparticular feature, structure, or characteristic described in connectionwith the example is included in at least one embodiment. Thus,appearances of the phrases “in an example” in various places throughoutthis specification are not necessarily all referring to the sameembodiment.

Example Embodiments

An initial overview of invention embodiments is provided below andspecific embodiments are then described in further detail. This initialsummary is intended to aid readers in understanding the technologicalconcepts more quickly, but is not intended to identify key or essentialfeatures thereof, nor is it intended to limit the scope of the claimedsubject matter.

Neurological disorders can stem from various causes, such as congenitalabnormalities, environmental factors, genetic disorders, infections,injury, lifestyle, and/or malnutrition, to name a few.

As one example, autosomal dominant cerebellar ataxias (ADCAs) are aheterogeneous group of neurodegenerative disorders characterized byprogressive degeneration of the cerebellum, brain stem, and spinal cord.Degeneration occurs at the cellular level and in certain subtypesresults in cellular death. Cellular death or dysfunction can interferewith the line of communication from the central nervous system to targetmuscles in the body.

Clinically, ADCA has been divided into three types (ADCA types I, II,and III), which are further divided into various sub-types. Each ofthese types and sub-types can cause a variety of deleterious effects.Type I ADCA, sub-type 1 conditions are typically characterized by CAGnucleotide repeats in the DNA, which code for polyglutamine. Thesepolyglutamine segments can cause degenerative effects on the proteinlevel. As non-limiting examples, ADCA Type I, sub-type 1 can includeSCA1, SCA2, SCA3, SCA17, and DRPLA, among others. However, manyneurodegenerative disorders can be associated with other trinucleotiderepeat expansions, or other genetic repeat expansions, besides CAGnucleotide repeats.

As one illustrative example of a neurodegenerative disorder,spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant cerebellarataxia characterized by progressive degeneration of cerebellar Purkinjecells (PCs), and selective loss of neurons within the brainstem andspinal cord. As described above, the genetic defect in SCA2 is expansionof CAG repeats (code for polyglutamine) in exon 1 of ATN2 gene. Mutantataxin-2 (ATXN2) protein with excessive polyglutamine acquires gain offunction and shows aggregation in the SCA2 brain. ATXN2 aggregates anddegeneration of cerebellar PCs, and altered RNA expressions arepathological consequences of expanded CAG repeat expression in SCA2cells.

ATXN2 is widely expressed in the mammalian nervous system. ATXN2 isinvolved in regulation of the EGF receptor, inositol 1,4,5-triphosphatereceptor (IP3R), and has role in translational regulation as well asembryonic development. ATXN2 interacts with multiple RNA bindingproteins, including RNA splicing factor A2BP1/Fox1, polyA bindingprotein 1 (PABP1), DDX6, and Tar DNA binding protein-43 (TDP-43)demonstrating its unique role in RNA metabolism. Furthermore, ATXN2 is aconstituent protein of subcellular components, like stress granules(SGs) and P-bodies, supporting its function in sequestering mRNAs andregulating protein translation during stress.

Further, double-stranded RNA-binding proteins Staufen1 (STAU1) andstaufen-2 (STAU2) can be recruited to cytoplasmic inclusions in brainoligodendrocytes and other cultured cells and modulate SGs dynamics.Staufen1 can perform a number of RNA-related functions such as mRNAtransport and degradation as well as being a protein localized to RNAgranules under cellular stress. Although Staufen1 can be associated withmRNA transport in both oocytes and somatic cells in vertebrates,Staufen1 is a multifunctional protein involved in regulating RNAmetabolism in various cell types. Furthermore, Staufen1 can regulate thetranslational efficiency of a population of mRNAs via 5′UTR and thestability of transcripts through 3′UTR, a mechanism referred to asStaufen-mediated RNA decay (SMD).

It is noted that RNA metabolism, including translation, transport,storage, and degradation, have assumed important roles in cellularfunction both in health and disease. Stress granules play an importantrole in RNA metabolism as they can regulate translation in times ofcellular stress, and RNA granules can regulate mRNA transport and localexpression control. SGs are composed of mRNA, ribosomal RNAs, andnon-coding RNAs as well as a specific complement of proteins that canvary from cell to cell.

With this in mind, ATXN2 can interact with multiple RNA bindingproteins, including PABP1, A2BP1/Fox1, DDX6 and TDP-43, suggesting aunique role for ATXN2 in RNA metabolism. Further, Staufen1 is aRNA-dependent interactor for ATXN2. Although the strength ofprotein-protein interaction does not appear to differ for wild-type andmutant ATXN2, in the presence of mutant ATXN2, steady-state Staufen1levels can be greatly increased in SCA2 patient-derived cells. Thus, aswill be described in further detail herein infra, in some cases, theexpansion of the polyglutamine regions in the mutant ATXN2 protein, orother proteins having a polynucleotide expansion, can facilitateincreased levels of Staufen1 in SCA2 patient-derived cells. Furtherstill, increased levels of Staufen1 in nerve cells can play an importantrole in the deleterious effects of ADCA (e.g. ADCA type I, sub-type 1,etc.) and other neurodegenerative disorders.

As some additional examples, human chromosome 9 open reading frame 72(c9orf72) is a protein endcoded by the gene C9orf72. However, mutationsin C9orf72 have been identified to be a genetic link between at leastfamilial frontotemporal demential (FTD) and amyotrophic lateralsclerosis (ALS). In further detail, the mutation of C9orf72 is ahexanucleotide repeat expansion of the six letter string of nucleotidesGGGGCC. Normally, there are less than about 20-30 of thesehexanucleotide repeats present in the C9orf72 gene. However, where aC9orf72 mutation is present, this hexanucleotide repeat can occur at afrequency of greater than 30 repeats (e.g. greater than 100 repeats, forexample). This mutation can interfere with the normal expression ofC9orf72 protein.

Also, TAR DNA-binding protein 43 (TDP-43) is a protein encoded by theTARDBP gene. However, mutations in TARDBP gene and/or TDP-43 can causepathogenic forms of TDP-43, which can be associated with a number ofneurological disorders, such as certain forms of frontotemporaldementia, ALS, chronic traumatic encephalopathy, certain subsets ofAlzheimers disease, among others.

Additionally, Tau proteins are the product of alternative splicing froma single gene designated as microtubule-associated protein tau (MAPT).However, certain mutations in MAPT and/or Tau proteins can result inpathogenic forms of Tau, which can be associated with a number ofneurological disorders, such as Alzheimers disease, frontotemporaldementia, and a variety of other neurological disorders.

Further, Amyloid precursor protein (APP) is an integral membrane proteinconcentrated in the synapses of neurons. However, mutations in APPand/or APP can be associated with familial susceptibility to Alzheimersdisease.

Thus, a variety of mutations can be involved in neurological disorders.However, as non-limiting examples, Staufen1 levels can be increased incells with the CAG repeat expansions in AXTN2, in cells with thehexanucleotide repeat expansions in C9orf72, in cells with TDP-43mutations, in cells with MAPT mutations, and in cells with APPmutations. This is additional evidence that Staufen1 can be a valuabletherapeutic target in the treatment of a variety of neurologicaldisorders, whether Staufen1 is targeted directly or indirectly, as willbe described in further detail herein.

Accordingly, the present disclosure describes compositions and methodsfor normalizing or controlling Staufen1 levels or activity in a targetcell (e.g. a nerve cell). As one non-limiting example, a method ofminimizing dysregulation of Staufen1-associated RNA metabolism isdescribed. The method can include introducing an amount of aStaufen1-regulating agent to a target cell sufficient to minimize thedysregulation.

A method of controlling Staufen1 activity is also described. The methodcan include introducing an amount of a Staufen1-regulating agent to atarget cell sufficient to reduce Staufen1 activity as compared toStaufen1 activity prior to or without introducing theStaufen1-regulating agent.

Further, a method of controlling Staufen1 accumulation in a target cellis described. The method can include introducing an amount of a Staufen1regulating agent to a target cell sufficient to reduce a concentrationof Staufen1 in the target cell as compared to the concentration in thetarget cell prior to or without introducing the Staufen1-regulatingagent.

Further still, a method of treating a neurodegenerative conditionassociated with Staufen1-induced dysregulation of RNA metabolism isdescribed. The method can include administering a therapeuticallyeffective amount of a Staufen1-regulating agent to a target cell of asubject.

A therapeutic agent or composition for treating a neurodegenerativecondition associated with Staufen1-induced dysregulation of RNAmetabolism is also described. The therapeutic agent can be aStaufen1-regulating agent. The therapeutic composition can include atherapeutically effective amount of a Staufen1-regulating agent and apharmaceutically acceptable carrier.

In the present disclosure, it is noted that when discussing the variousmethods, the therapeutic agent, and the therapeutic composition, each ofthese discussions can be considered applicable to each of theseexamples, whether or not they are explicitly discussed in the context ofthat example. Thus, for example, in discussing details about a methodper se, such discussion also refers to the other methods describedherein, the therapeutic agent, the therapeutic composition, therapeuticdosage amounts and forms, and vice versa.

In further detail, the present disclosure describes methods ofminimizing dysregulation of Staufen1-associated RNA metabolism. Asdescribed above, Staufen1 can perform a number of RNA-related functions,such as mRNA transport and degradation, for example. Thus, in somecases, having elevated Staufen1 levels in a target cell can lead todysregulation of Staufen1-associated RNA metabolism. Further, in someexamples, elevated levels of Staufen1 in a target cell can lead toundesired and accelerated degradation of various cellular components.Accordingly, the methods can include introducing a sufficient amount ofa Staufen1-regulating agent to a target cell to minimizeStaufen1-associated dysregulation of RNA metabolism.

A variety of Staufen1-regulating agents can be employed in the methodsrecited herein. It is noted that when discussing Staufen1-regulatingagents per se, it is to be understood that a Staufen1-regulating agentcan act or function to regulate Staufen1 without directly interactingwith Staufen1. For example, in some cases a Staufen1-regulating agentcan be an agent that acts upstream from Staufen1 to regulate Staufen1.As one non-limiting example, in some cases, a Staufen1-regulating agentcan be a therapeutic agent that inhibits expression of mutant ATXN2. Inthis way, the staufen1-regulating agent can facilitate regulation ofStaufen1 without directly interacting with Staufen1 protein or STAU1gene, but rather with cellular components or genes which have a director indirect impact on Staufen1 protein, including its properties, orbehavior, such as lifespan, residence time, binding availability,stability, cellular accumulation, etc. Thus, in some examples, aneffective Staufen1-regulating agent can also include an gene therapyagent, such as for modifying a mutant allele to a wild-type allele tofacilitate regulation of Staufen1.

As non-limiting examples, in some cases, the Staufen1-regulating agentcan be or include a Staufen1-inhibiting agent. A Staufen1-inhibitingagent can include any agent that fully or partially inhibits thetranscription or translation of Staufen1 in the target cell. In someadditional examples, the Staufen1-regulating agent can be or include aStaufen1-inactivating agent. A Staufen1-inactivating agent can includeany agent that binds to, degrades, etc. the Staufen1 protein so as toreduce or eliminate its RNA metabolism (e.g. RNA transportation, RNAdegradation, etc.) function in the target cell. In some examples, theStaufen1-regulating agent can reduce or block Staufen1 interaction withmRNAs, which can result in altered mRNA expression or abundance.

In some examples, the Staufen1-regulating agent can be or include amutant ATXN2-inhibiting agent (or inhibiting agent for another mutantgene having excessive nucleotide repeats). A mutant ATXN2-inhibitingagent can be any agent that inhibits the transcription or translation ofmutant ATXN2 (e.g. ATXN2 having 32 or more polyglutamine repeats) in thetarget cell. In some examples, the mutant ATXN2-inhibiting agent can beor include an agent that can partially or fully silence transcription ortranslation of mutant ATXN2. In some additional examples, theATXN2-inhibiting agent can be or include an agent that can edit themutant ATN2 gene to modify the mutant allele to a wild-type sequence(e.g. to include from about 21 to about 31, from about 21 to about 25,or from about 22 to about 23 polyglutamine repeats, rather than 32 ormore). As will be recognized by one skilled in the art, otherneurodegenerative diseases can be affected by excessive polynucleotiderepeats on genes other than ATXN2 where the normal number of repeats canvary from the ranges listed above. In such cases, the mutant gene can beedited to include a polynucleotide repeat within the wild-type range. Insome examples, the Staufen1-regulating agent can be or include a mutantATXN2-inactivating agent. The mutant ATXN2-inactivating agent caninclude any agent that binds to, degrades, or otherwise inactivates themutant ATXN2 protein (or another mutant protein having excessivenucleotide repeats) or prevents or reduces interaction of the mutantATXN2 protein with Staufen1 in a manner that minimizes excessiveaccumulation of Staufen1 in the target cell.

For example, in some cases, the Staufen1-regulating agent can be orinclude a mutant c9orf72-inhibiting agent (or inhibiting agent foranother mutant gene having excessive nucleotide repeats). A mutantc9orf72-inhibiting agent can be any agent that inhibits thetranscription or translation of mutant c9orf72 (e.g. c9orf72 havinggreater than 30 GGGGCC hexanucleotide repeats (e.g. greater than 100GGGGCC hexanucleotide repeats, for example)) in the target cell. In someexamples, the mutant c9orf72-inhibiting agent can be or include an agentthat can partially or fully silence transcription or translation ofmutant c9orf72. In some additional examples, the c9orf72-inhibitingagent can be or include an agent that can edit the mutant C9orf72 geneto modify the mutant allele to a wild-type sequence (e.g. to includeabout 20-30 GGGGCC hexanucleotide repeats or less). In some examples,the Staufen1-regulating agent can be or include a mutantc9orf72-inactivating agent. The mutant c9orf72-inactivating agent caninclude any agent that binds to, degrades, or otherwise inactivates themutant c9orf72 protein (or another mutant protein having excessivenucleotide repeats) or prevents or reduces interaction of the mutantc9orf72 protein with Staufen1 in a manner that minimizes excessiveaccumulation of Staufen1 in the target cell.

In some additional examples, the Staufen1-regulating agent can be orinclude a mutant TDP-43-inhibiting agent. A mutant TDP-43-inhibitingagent can be any agent that inhibits the transcription or translation ofmutant TDP-43 in the target cell. In some examples, the mutantTDP-43-inhibiting agent can be or include an agent that can partially orfully silence transcription or translation of mutant TDP-43. In someadditional examples, the TDP-43-inhibiting agent can be or include anagent that can edit the mutant TARDBP gene to modify the mutant alleleto a wild-type sequence. In some examples, the Staufen1-regulating agentcan be or include a mutant TDP-43-inactivating agent. The mutantTDP-43-inactivating agent can include any agent that binds to, degrades,or otherwise inactivates the mutant TDP-43 protein or prevents orreduces interaction of the mutant TDP-43 protein with Staufen1 in amanner that minimizes excessive accumulation of Staufen1 in the targetcell.

In some further examples, the Staufen1-regulating agent can be orinclude a mutant Tau-inhibiting agent. A mutant Tau-inhibiting agent canbe any agent that inhibits the transcription or translation of mutantTau in the target cell. In some examples, the mutant Tau-inhibitingagent can be or include an agent that can partially or fully silencetranscription or translation of mutant Tau. In some additional examples,the Tau-inhibiting agent can be or include an agent that can edit themutant MAPT gene to modify the mutant allele to a wild-type sequence. Insome examples, the Staufen1-regulating agent can be or include a mutantTau-inactivating agent. The mutant Tau-inactivating agent can includeany agent that binds to, degrades, or otherwise inactivates the mutantTau protein or prevents or reduces interaction of the mutant Tau proteinwith Staufen1 in a manner that minimizes excessive accumulation ofStaufen1 in the target cell.

In yet other examples, the Staufen1-regulating agent can be or include amutant APP-inhibiting agent. A mutant APP-inhibiting agent can be anyagent that inhibits the transcription or translation of mutant APP inthe target cell. In some examples, the mutant APP-inhibiting agent canbe or include an agent that can partially or fully silence transcriptionor translation of mutant APP. In some additional examples, theAPP-inhibiting agent can be or include an agent that can edit the mutantAPP gene to modify the mutant allele to a wild-type sequence. In someexamples, the Staufen1-regulating agent can be or include a mutantAPP-inactivating agent. The mutant APP-inactivating agent can includeany agent that binds to, degrades, or otherwise inactivates the mutantAPP protein or prevents or reduces interaction of the mutant APP proteinwith Staufen1 in a manner that minimizes excessive accumulation ofStaufen1 in the target cell.

It is noted that it may not always be desirable to inhibit theexpression of certain mutant genes where only one copy of the gene ismutated and the gene is functionally important to produce a wild-typephenotype. For example, in some cases, a single functional copy of agene may be sufficient to produce a wild-type phenotype. In contrast,inhibiting the expression of the mutant gene may also inhibit theexpression of the unmutated gene, which can cause insufficientexpression to produce the wild-type phenotype. However, where this is aconcern, in some cases it may be possible to express the wild-typeallele via an exogenous expression vector in an amount sufficient toproduce the wild-type phenotype where inhibition of the mutant gene isdesirable.

In some examples, the Staufen1-regulating agent can be a mechanistictarget of rapamycin (mTOR)-inhibiting agent. An mTOR-inhibiting agentcan be any agent that inhibits the transcription or translation of mTORin the target cell. In some further examples, the Staufen1-regulatingagent can be an mTOR-inactivating agent. An mTOR inactivating agent canbe any agent that can bind to, degrade, etc. mTOR protein to prevent orminimize its interaction with Staufen1 protein.

Thus, there are a variety of Staufen1-regulating agents that can beemployed to perform one or more of these functions. Non-limitingexamples of Staufen1-regulating agents can include siRNAs, miRNAs,antisense oligonucleotides (with chemistries including phosphorothioatebase pair linkages, 2′-methoxyethyl, 2′-O-methyl, 2′-fluoro,2′-(3-hydroxy)-propyl, locked nucleic acid (LNA), peptide nucleic acid(PNA), cyclohexene nucleic acid (CeNA), hexitol nucleic acids (HNA),morpholino, in combinations including mixed, chimeric or gapmer),ribozymes, peptide nucleic acids, morpholinos, small molecules, thelike, or combinations thereof.

In some specific examples, the Staufen1-regulating agent can be anantisense oligonucleotide (ASO). In some aspects, the ASO can includeone or more suitable modifications, such as sulfur for oxygensubstitutions (e.g. introduction of phosphorothioate linkages), 2′-OHmodifications, 2′-O-methyl modifications, 2′-fluoro modifications,2′-O-methoxyethyl modifications, locked nucleic acid (LNA)modifications, bridged nucleic acid (BNA) modifications, peptide nucleicacid (PNA) modifications, morpholino modifications, hexitol nucleic acid(HNAs) modifications, introduction of central phosphodiester orphosphorothioate residues (e.g. to form a “gapmer”), the like, orcombinations thereof.

In some examples, the ASO can be a gapmer, such as an A_(x)-G_(n)-A_(x)gapmer, where A represents an artificial nucleotide monomer and x is aninteger from 2 to 7, and where G represents the gap or block of naturalantisense nucleotide monomers and n is an integer from 8 to 12. Wherethe ASO is a gapmer, a variety of artificial nucleotide monomers can beused in the 5′ and 3′ wings, such as those mentioned above. For example,non-limiting examples can include 2-O-methoxyethyl (MOE), 2′,4-bridgednucleic acid (BNA), locked nucleic acid (LNA), N-Me-aminooxy BNA,2′-N-(methyl)-4-C-aminooxy methylene 2′,4-bridged nucleic acid,N-Me-aminooxy′ 2′,4-bridged nucleic acid,2′-N-(methyl)-4′C-aminooxymetylene 2%4′-bridged nucleic acid,2′,4′BNA^(NC)[NMe], 2′-O,4′C-(N-methyl) aminomethylene, 2′,4′-bridgednucleic acid, 2′,4′BNA^(NC)2′-O,4′-C-aminomethylene 2′,4-bridged nucleicacid, the like, or a combination thereof. In some specific examples, thegapmer can be a MOE gapmer. In other specific examples, the gapmer canbe an LNA gapmer. In some further examples, the gapmer can be a 5-8-5gapmer, a 5-10-5 gapmer, or a 5-12-5 gapmer. In some additionalexamples, the gapmer can be a 4-8-4 gapmer, a 4-10-4 gapmer, or a 4-12-4gapmer. Instill additional examples, the gapmer can be a 3-8-3 gapmer, a3-10-3 gapmer or a 3-12-3 gapmer

Table 1 presents some non-limiting examples of ASOs that can be includedin a therapeutic composition as described herein.

SEQ ID NO: Antisense Sequence (5′ to 3′) SEQ ID NO: 1TCTCATGTTGTAGTTATAGG SEQ ID NO: 2 CTGGAAAGATAGTCCAGTTG SEQ ID NO: 3TCGGGCTATCATGGCAGTTA SEQ ID NO: 4 CTCGGGCTATCATGGCAGTT SEQ ID NO: 5TCTCGGGCTATCATGGCAGT SEQ ID NO: 6 CTCTCGGGCTATCATGGCAG SEQ ID NO: 7GCTGTGGGCGAGGTGCCCCC SEQ ID NO: 8 CGGCTGTGGGCGAGGTGCCC SEQ ID NO: 9CATGGCAGTTACCGTTGCCT SEQ ID NO: 10 GCTATCATGGCAGTTACCGT SEQ ID NO: 11AACTCTCGGGCTATCATGGC SEQ ID NO: 12 TACAACAACTCTCGGGCTAT SEQ ID NO: 13TAAACTCTGGCTGCTGCTCC SEQ ID NO: 14 AGTGGTTAATAAACTCTGGC SEQ ID NO: 15GTTCTGAGAGGTTAAGTGGT SEQ ID NO: 16 TTTGTTCAGTTCTGAGAGGT SEQ ID NO: 17TTCCAGGAACAATGTTGTCT SEQ ID NO: 18 AAACAGCTTTCAGTGCAGGT SEQ ID NO: 19ACAAATGCAGGTAAACAGCT SEQ ID NO: 20 AGACATGGTCACTTTCAACA SEQ ID NO: 21CACTTGAACTTGAGACATGG SEQ ID NO: 22 TTCTGAACTTGCACTTGAAC SEQ ID NO: 23TCAGTATTTGGCTCCCTGAG SEQ ID NO: 24 TCTTGTTCAGTATTTGGCTC SEQ ID NO: 25GCTGTGAGAGAAGAGACTGG SEQ ID NO: 26 GGTCTACCTGCATTTTCAGA SEQ ID NO: 27AGCTGCACTGGTGGATGTAA SEQ ID NO: 28 CACAGTGCATTTAGTTCTAC SEQ ID NO: 29TCATGCACAGTGCATTTAGT SEQ ID NO: 30 CCAAGTTTCATGCACAGTGC SEQ ID NO: 31CAACAGGCTTATACATTGGT SEQ ID NO: 32 AGTTATAGGTGGACTGCATC SEQ ID NO: 33CCCACAGAAAGTTCCACTTG SEQ ID NO: 34 TGCCATTAAATTGCTGTCCT SEQ ID NO: 35GTCTTGTCTTTCCTTTGCCA SEQ ID NO: 36 GCAGCCTGTCTTGTCTTTCC SEQ ID NO: 37GTGCAATCTCAAACACTTGA SEQ ID NO: 38 TGGTCACAAAGTTCTTCATG SEQ ID NO: 39TGGTGTGATGTCCTTGACTA SEQ ID NO: 40 GGTTCAGCACCTCCCACACA

In some non-limiting examples, the ASO can be or include a nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, or a combination thereof. In someadditional examples, the ASO can be or include one or more nucleotidesequences that are at least 95% homologous to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, orthe middle 8, 10, or 12 nucleotides thereof. In still additionalexamples, the ASO can be or include one or more nucleotide sequencesthat are at least 90% homologous with SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, or the middle8, 10, or 12 nucleotides thereof. In some further examples, the ASO canbe or include one or more nucleotide sequences that are at least 85%homologous with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, or the middle 8, 10, or 12nucleotides thereof. In some further examples, the ASO can be or includeone or more nucleotide sequences that are at least 80% homologous withSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, or SEQ ID NO: 40, or the middle 8, 10, or 12 nucleotidesthereof.

In some examples, the ASO can be or include one or more nucleotidesequences having at least 6 consecutive nucleotides of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO:40. In some examples, the 6 consecutive nucleotides can be the 6 middlenucleotides of the listed nucleotide sequences. In some additionalexamples, the ASO can be or include one or more nucleotide sequenceshaving at least 8 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. Insome examples, the 8 consecutive nucleotides can be the 8 middlenucleotides of the listed nucleotide sequences. In some additionalexamples, the ASO can be or include one or more nucleotide sequenceshaving at least 10 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. Insome examples, the 10 consecutive nucleotides can be the 10 middlenucleotides of the listed nucleotide sequences. In some additionalexamples, the ASO can be or include one or more nucleotide sequenceshaving at least 12 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. Insome examples, the 12 consecutive nucleotides can be the 12 middlenucleotides of the listed nucleotide sequences. In some additionalexamples, the ASO can be or include one or more nucleotide sequenceshaving at least 14 consecutive nucleotides of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. Insome examples, the 14 consecutive nucleotides can be the 14 middlenucleotides of the listed nucleotide sequences.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 1. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 1. In some examples, the consecutive nucleotides of SEQ ID NO: 1 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 1. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 1 caninclude the middle 10 nucleotides (i.e., TGTTGTAGTT). In some examples,the ASO can be or include a nucleotide sequence that is at least 90%,80%, or 70% homologous to the middle 10 nucleotides of SEQ ID NO: 1.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%,90%, 85%, or 80% homologous to SEQ ID NO: 2. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 2. In some examples, the consecutive nucleotides of SEQ ID NO: 2 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 2. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 2 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 2.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%,85%, or 80% homologous to SEQ ID NO: 3. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 3. In some examples, the consecutive nucleotides of SEQ ID NO: 3 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 3. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 3 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 3.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%,90%, 85%, or 80% homologous to SEQ ID NO: 4. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 4. In some examples, the consecutive nucleotides of SEQ ID NO: 4 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 4. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 4 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 4.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 5. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 5. In some examples, the consecutive nucleotides of SEQ ID NO: 5 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 5. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 5 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 5.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 6. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 6. In some examples, the consecutive nucleotides of SEQ ID NO: 6 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 6. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 6 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 6.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 7. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 7. In some examples, the consecutive nucleotides of SEQ ID NO: 7 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 7. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 7 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 7.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 8. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 8. In some examples, the consecutive nucleotides of SEQ ID NO: 8 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 8. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 8 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 8.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%,90%, 85%, or 80% homologous to SEQ ID NO: 9. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 9. In some examples, the consecutive nucleotides of SEQ ID NO: 9 caninclude the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 9. Insome specific examples, the consecutive nucleotides of SEQ ID NO: 9 caninclude the middle 10 nucleotides. In some examples, the ASO can be orinclude a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 9.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 10. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 10. In some examples, the consecutive nucleotides of SEQ ID NO: 10can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 10.In some specific examples, the consecutive nucleotides of SEQ ID NO: 10can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 10.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%,85%, or 80% homologous to SEQ ID NO: 11. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 11. In some examples, the consecutive nucleotides of SEQ ID NO: 11can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 11.In some specific examples, the consecutive nucleotides of SEQ ID NO: 11can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 11.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 12. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 12. In some examples, the consecutive nucleotides of SEQ ID NO: 12can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 12.In some specific examples, the consecutive nucleotides of SEQ ID NO: 12can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 12.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90% 85%, or 80% homologous to SEQ ID NO: 13. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 13. In some examples, the consecutive nucleotides of SEQ ID NO: 13can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 13.In some specific examples, the consecutive nucleotides of SEQ ID NO: 13can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80% or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 13.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%, 85%, or 80% homologous to SEQ ID NO: 14. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 14. In some examples, the consecutive nucleotides of SEQ ID NO: 14can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 14.In some specific examples, the consecutive nucleotides of SEQ ID NO: 14can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 14.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 15. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 15. In some examples, the consecutive nucleotides of SEQ ID NO: 15can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 15.In some specific examples, the consecutive nucleotides of SEQ ID NO: 15can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 15.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 16. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 16. In some examples, the consecutive nucleotides of SEQ ID NO: 16can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 16.In some specific examples, the consecutive nucleotides of SEQ ID NO: 16can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%. 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 16.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 17. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 17. In some examples, the consecutive nucleotides of SEQ ID NO: 17can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 17.In some specific examples, the consecutive nucleotides of SEQ ID NO: 17can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 17.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 18. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 18. In some examples, the consecutive nucleotides of SEQ ID NO: 18can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 18.In some specific examples, the consecutive nucleotides of SEQ ID NO: 18can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%. 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 18.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 19. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 19. In some examples, the consecutive nucleotides of SEQ ID NO: 19can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 19.In some specific examples, the consecutive nucleotides of SEQ ID NO: 19can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80% or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 19.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%, 85%, or 80% homologous to SEQ ID NO: 20. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 20. In some examples, the consecutive nucleotides of SEQ ID NO: 20can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 20.In some specific examples, the consecutive nucleotides of SEQ ID NO: 20can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 20.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 21. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 21. In some examples, the consecutive nucleotides of SEQ ID NO: 21can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 21.In some specific examples, the consecutive nucleotides of SEQ ID NO: 21can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 21.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%, 85%, or 80% homologous to SEQ ID NO: 22. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 22. In some examples, the consecutive nucleotides of SEQ ID NO: 22can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 22.In some specific examples, the consecutive nucleotides of SEQ ID NO: 22can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 22.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 23. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 20. In some examples, the consecutive nucleotides of SEQ ID NO: 23can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 23.In some specific examples, the consecutive nucleotides of SEQ ID NO: 23can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 23.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%,85%, or 80% homologous to SEQ ID NO: 24. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 24. In some examples, the consecutive nucleotides of SEQ ID NO: 24can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 24.In some specific examples, the consecutive nucleotides of SEQ ID NO: 24can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80% or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 24.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%, 85%, or 80% homologous to SEQ ID NO: 25. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 25. In some examples, the consecutive nucleotides of SEQ ID NO: 25can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 25.In some specific examples, the consecutive nucleotides of SEQ ID NO: 25can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 25.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 26. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 26. In some examples, the consecutive nucleotides of SEQ ID NO: 26can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 26.In some specific examples, the consecutive nucleotides of SEQ ID NO: 26can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 26.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 27. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 27. In some examples, the consecutive nucleotides of SEQ ID NO: 27can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 27.In some specific examples, the consecutive nucleotides of SEQ ID NO: 27can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 27.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 28. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 28. In some examples, the consecutive nucleotides of SEQ ID NO: 28can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 28.In some specific examples, the consecutive nucleotides of SEQ ID NO: 28can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 28.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 29. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 29. In some examples, the consecutive nucleotides of SEQ ID NO: 29can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 29.In some specific examples, the consecutive nucleotides of SEQ ID NO: 29can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%. 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 29.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90% 85%, or 80% homologous to SEQ ID NO: 30. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 30. In some examples, the consecutive nucleotides of SEQ ID NO: 30can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 30.In some specific examples, the consecutive nucleotides of SEQ ID NO: 30can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80% or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 30.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%, 85%, or 80% homologous to SEQ ID NO: 31. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 31. In some examples, the consecutive nucleotides of SEQ ID NO: 31can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 31.In some specific examples, the consecutive nucleotides of SEQ ID NO: 31can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 31.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 32. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 32. In some examples, the consecutive nucleotides of SEQ ID NO: 32can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 32.In some specific examples, the consecutive nucleotides of SEQ ID NO: 32can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 32.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%,85%, or 80% homologous to SEQ ID NO: 33. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 33. In some examples, the consecutive nucleotides of SEQ ID NO: 33can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 33.In some specific examples, the consecutive nucleotides of SEQ ID NO: 33can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 33.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 34. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 34. In some examples, the consecutive nucleotides of SEQ ID NO: 34can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 34.In some specific examples, the consecutive nucleotides of SEQ ID NO: 34can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 34.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%85%, or 80% homologous to SEQ ID NO: 35. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 35. In some examples, the consecutive nucleotides of SEQ ID NO: 35can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 35.In some specific examples, the consecutive nucleotides of SEQ ID NO: 35can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80% or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 35.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%. 90%, 85%, or 80% homologous to SEQ ID NO: 36. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 36. In some examples, the consecutive nucleotides of SEQ ID NO: 36can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 36.In some specific examples, the consecutive nucleotides of SEQ ID NO: 36can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 36.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 37. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 37. In some examples, the consecutive nucleotides of SEQ ID NO: 37can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 37.In some specific examples, the consecutive nucleotides of SEQ ID NO: 37can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 37.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 38. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 38. In some examples, the consecutive nucleotides of SEQ ID NO: 38can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 38.In some specific examples, the consecutive nucleotides of SEQ ID NO: 38can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 38.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90% 85%, or 80% homologous to SEQ ID NO: 39. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 39. In some examples, the consecutive nucleotides of SEQ ID NO: 39can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 39.In some specific examples, the consecutive nucleotides of SEQ ID NO: 39can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 39.

In some examples, the ASO can be or include a nucleotide sequence thatis at least 95%, 90%, 85%, or 80% homologous to SEQ ID NO: 40. In someadditional examples, the ASO can be or include a nucleotide sequencehaving at least 6, 8, 10, 12, or 14 consecutive nucleotides of SEQ IDNO: 40. In some examples, the consecutive nucleotides of SEQ ID NO: 40can include the middle 6, 8, 10, 12, or 14 nucleotides of SEQ ID NO: 40.In some specific examples, the consecutive nucleotides of SEQ ID NO: 40can include the middle 10 nucleotides. In some examples, the ASO can beor include a nucleotide sequence that is at least 90%, 80%, or 70%homologous to the middle 10 nucleotides of SEQ ID NO: 40.

In some additional specific examples, the Staufen1-regulating agent canbe a small interfering RNA (siRNA). In some examples, the siRNA caninclude one or more suitable modifications, such as a 2′-sugarmodification, an altered ring structure, a nucleobase modification, thelike, or a combination thereof. It is further noted that the siRNA caninclude any suitable 3′ nucleotide overhangs. In some specific examples,the siRNA can include a nucleotide sequence of5-CCUAUAACUACAACAUGAGdTdT-3′ (SEQ ID NO: 41), 5′-GAGCCUUGUUGAUCCUUAA-3′(SEQ ID NO: 42), etc. on the guide/antisense strand. In some furtherexamples, the siRNA can include a nucleotide sequence that is at least95%, 90%, 85%, or 80% homologous to SEQ ID NO: 41 on the guide/antisensestrand. In some other examples, the siRNA can include a nucleotidesequence that has at least 10, 12, 14, 16, or 18 consecutive nucleotidesof SEQ ID NO: 41. In some further examples, the siRNA can include anucleotide sequence that is at least 95%. 90%, 85%, or 80% homologous toSEQ ID NO: 42. In some other examples, the siRNA can include anucleotide sequence that has at least 10, 12, 14, 16, or 18 consecutivenucleotides from SEQ ID NO: 42.

In some examples, the Staufen1-regulating agent can be a gene therapyagent that does not directly interact with Staufen1 protein or STAU1gene (e.g. a mutant ATXN2-inhibiting agent, a mutant c9orf72-inhibitingagent, a mutant TDP-43-inhibiting agent, a mutant Tau-inhibiting agent,and/or a mutant APP-inhibiting agent, for example). In such examples,the Staufen1-regulating agent can be used for gene therapy (e.g.homologous recombination, CRISPR/Cas9 gene editing, etc.) to permanentlyalter the DNA to prevent expression of mutant ATXN2 (or other similargene including excessive CAG repeats or other polynucleotide repeats),mutant C9orf72, mutant TARDBP, mutant MAPT, and/or mutant APP. In somespecific examples, CRISPR/Cas9 systems can be employed. For example, bydelivering a Cas9 nuclease complexed with a synthetic guide RNA into atarget cell, the cell's genome can be cut at a desired location,allowing existing genes to be removed and/or altered genes to be added.Thus, in some examples, a CRISPR/Cas9 system can be administered to anindividual having a neurodegenerative disorder to remove or edit themutant ATXN2 gene mutant C9orf72 gene, mutant TARDBP gene, mutant MAPTgene, and/or mutant APP gene and replace it with the wild-type versionof the gene.

The Staufen1-regulating agent can be introduced to the target cell in avariety of ways. In some examples, the Staufen1-regulating agent can beintroduced to the target cell as a “free” Staufen1-regulating agent thatis not bound to or carried by a delivery vector or vehicle. However, aswill be recognized by one skilled in the art, there are a number ofchallenges to in vivo delivery of certain types of therapeutic agents.As such, in other examples, the Staufen1-regulating agent can beintroduced to the target cell via a delivery vector or vehicle. Forexample, in some cases, the Staufen1-regulating agent can be bound to orcarried by a cell targeting agent that targets a specific cell surfacereceptor to facilitate introduction to the target cell. Such celltargeting agents can include aptamers, antibodies/fragments,polypeptides (e.g. polypeptides derived from phage display libraries),N-acetylgalactosamine, vitamins, small organic molecules, the like, or acombination thereof. In some examples, the Staufen1-regulating agent canbe bound to or carried by a cell penetration agent to facilitatetransmembrane permeation of the Staufen1-regulating agent at the targetcell. Such cell penetration agents can include polycationic peptides(e.g. polycationic peptides rich in argine and/or lysine, other cellpenetrating peptides, etc.), polymers (e.g. PEGylated polycations,polyethyleneimine (PEI), cationic block co-polymers, etc.), dendrimers(e.g. octaguanidine dendrimers, PEI-based dendrimers, etc.), lipids(e.g. cationic lipids, cholesterol, etc.), lysosomal carriers,liposomes, micelles, quantum dots, nanoparticles, the like, or acombination thereof. In still other examples, the Staufen1-regulatingagent can be introduced to the target cell via a viral vector.Non-limiting examples of viral vectors that can be employed can includeretrovirus, lentivirus, cytomegalovirus, adenovirus, adeno-associatedvirus, or a combination thereof. In some further examples, a viralvector can be used for long-term expression of the Staufen1-regulatingagent in the target cell. Thus, in some examples, theStaufen1-regulating agent can be introduced to a target cell via a viralvector. In other examples, the Staufen1-regulating agent can beintroduced to a target cell via a non-viral vector. In still otherexamples, the Staufen1-regulating agent can be introduced to the targetcell without a delivery vector or vehicle. In some further examples, anysuitable combination thereof can be employed.

The amount of Staufen1-regulating agent introduced to a target cell candepend on a variety of factors, such as the particularStaufen1-regulating agent being employed, the type and severity of thecondition, the dosing regimen, the type of delivery vector (whereemployed), etc., as will be appreciated by one skilled in the art. Thus,it can be more effective to describe the amount of Staufen1-regulatingagent introduced to a target cell by the effect of the amount on thetarget cell. For example, an effective amount of Staufen1-regulatingagent can restore/increase PCP2 mRNA levels in the target cell, CALB1mRNA levels in the target cell, other mRNAs in the target cell that aremetabolized via Staufen1-associated metabolism, or a combination thereofas compared to the levels of these mRNAs prior to or without introducingthe Staufen1-regulating agent. In some examples, a level of one or moreof these mRNAs can increase by at least 10%, 20%, 30%, 40% or more viaintroduction of the Staufen1-regulating agent as compared to the levelprior to or without introduction of the Staufen1-regulating agent.

In some examples, the amount of Staufen1-regulating agent introduced tothe target cell can reduce the amount of Staufen1 present in the targetcell as compared to the amount of Staufen1 present in the target cellprior to or without introduction of the Staufen1-regulating agent. Insome specific examples, the amount of Staufen1 present in the targetcell can be reduced by at least 30%, 40%, 50%, 60%, or 70% as comparedto Staufen1 levels prior to or without introduction of theStaufen1-regulating agent.

In still other examples, where the Staufen1-regulating agent is a mutantATXN2-inhibiting agent, the amount of Staufen1-regulating agent canreduce a mutant ATXN2 level (or a level of another protein including anelongated polyglutamine tract or similar expansion tract) in the targetcell as compared to the mutant ATXN2 level prior to or withoutadministration of the Staufen1-regulating agent. In some specificexamples, the amount of mutant ATXN2 (or other protein including anelongated polyglutamine tract or similar expansion tract) present in thetarget cell can be reduced by at least 30%, 40%, 50%, 60%, or 70% ascompared to mutant ATXN2 levels prior to or without introduction of theStaufen1-regulating agent.

In yet other examples, where the Staufen1-regulating agent is a mutantc9orf72-inhibiting agent, the amount of Staufen1-regulating agent canreduce a mutant c9orf72 level (or a level of another protein includingan elongated nucleotide expansion tract) in the target cell as comparedto the mutant c9orf72 level prior to or without administration of theStaufen1-regulating agent. In some specific examples, the amount ofmutant c9orf72 (or other protein including an elongated nucleotideexpansion tract) present in the target cell can be reduced by at least30%, 40%, 50%, 60%, or 70% as compared to mutant c9orf72 levels prior toor without introduction of the Staufen1-regulating agent.

In additional examples, where the Staufen1-regulating agent is a mutantTDP-43-inhibiting agent, the amount of Staufen1-regulating agent canreduce a mutant TDP-43 level in the target cell as compared to themutant TDP-43 level prior to or without administration of theStaufen1-regulating agent. In some specific examples, the amount ofmutant TDP-43 present in the target cell can be reduced by at least 30%,40%, 50%, 60%, or 70% as compared to mutant TDP-43 levels prior to orwithout introduction of the Staufen1-regulating agent.

In still other examples, where the Staufen1-regulating agent is a mutantTau-inhibiting agent, the amount of Staufen1-regulating agent can reducea mutant Tau level in the target cell as compared to the mutant Taulevel prior to or without administration of the Staufen1-regulatingagent. In some specific examples, the amount of mutant Tau present inthe target cell can be reduced by at least 30%, 40%, 50%, 60%, or 70% ascompared to mutant Tau levels prior to or without introduction of theStaufen1-regulating agent.

In still other examples, where the Staufen1-regulating agent is a mutantAPP-inhibiting agent, the amount of Staufen1-regulating agent can reducea mutant APP level in the target cell as compared to the mutant APPlevel prior to or without administration of the Staufen1-regulatingagent. In some specific examples, the amount of mutant APP present inthe target cell can be reduced by at least 30%, 40%, 50%, 60%, or 70% ascompared to mutant APP levels prior to or without introduction of theStaufen1-regulating agent.

The method of controlling, regulating, or normalizing Staufen1 activityin a target cell can include introducing an amount of aStaufen1-regulating agent to a target cell sufficient to reduce Staufen1activity as compared to Staufen1 activity prior to or withoutintroducing the Staufen1-regulating agent. It is noted that, in thisparticular method, it is not necessary to reduce the amount of Staufen1present in the target cell to control, normalize, or minimize Staufen1activity in the target cell. However, this is also one mechanism ofaccomplishing reduced Staufen1 activity in the target cell. In otherexamples, (e.g. via a mutant ATXN2-inactivating agent or mutantATXN2-inhibiting agent, or other upstream inactivating agent orinhibiting agent, such as those described above, for example) Staufen1activity can be reduced by reducing the mutant ATXN2 (or other proteinincluding an elongated polyglutamine tract or similar expansion tract,or other mutant protein as described above) present in the target cell.This can be accomplished by a number of methods, such as inhibitingexpression of the mutant ATXN2 gene (or other protein including anelongated polyglutamine tract or similar expansion tract, or othermutant gene associated with neurological disorders), editing the mutantATXN2 gene (or other protein including an elongated polyglutamine tractor similar expansion tract, or other mutant gene associated withneurological disorders, such as those described above) to a wild-typesequence, the like, or a combination thereof. In still other examples,the activity of Staufen1 can be reduced by introducing aStaufen1-regulating agent that can bind to, degrade, etc. Staufen1 toinactivate or otherwise interfere with the metabolic function ofStaufen1 in the target cell. In still other examples, theStaufen1-regulating agent can bind to, degrade, etc. mutant ATXN2 (orother protein including an elongated polyglutamine tract or similarexpansion tract, or other mutant protein associated with neurologicaldisorders, such as those described above) to minimize aggregation ofStaufen1 thereto, stabilization of Staufen1, the like, or a combinationthereof. In some additional examples, the Staufen1-regulating agent canreduce or block Staufen1 interaction with mRNAs, which can result inaltered mRNA expression or abundance.

The same types of Staufen1-regulating agents described herein supra canalso be employed in the method of controlling Staufen1 activity in atarget cell. Additionally, the same methods of introducing theStaufen1-regulating agent to the target cell as described herein supracan likewise be used in the method of controlling Staufen1 activity.Further, the same or similar amounts as described herein supra may beemployed in the method of controlling Staufen1 activity, depending onthe particular patient, type and severity of the condition, dosingregimen, Staufen1-regulating agent being employed, etc.

The method of controlling Staufen1 accumulation in a target cell caninclude introducing an amount of a Staufen1-regulating agent to a targetcell sufficient to reduce a concentration of Staufen1 in the target cellas compared to the concentration in the target cell prior to or withoutintroducing the Staufen1-regulating agent. In this particular method,the amount of accumulation of Staufen1 in the target cell can becontrolled, normalized, or minimized in a variety of ways. In someexamples, the accumulation of Staufen1 in the target cell can becontrolled by inhibiting Staufen1 expression. In some additionalexamples, controlling Staufen1 accumulation can be accomplished byreducing the mutant ATXN2 (or other protein including an elongatedpolyglutamine tract or similar expansion tract, or other mutant proteinassociated with neurological disorders, such as those described above)present in the target cell, such as by inhibiting expression of themutant ATXN2 gene (or other protein including an elongated polyglutaminetract or similar expansion tract, or other mutant protein associatedwith neurological disorders, such as those described above), editing themutant ATXN2 gene (or other gene including an elongated polyglutaminetract or similar expansion tract, or other mutant gene associated withneurological disorders, such as those described above) to a wild-typesequence, the like, or a combination thereof. In other examples,controlling Staufen1 accumulation can be accomplished by binding to,degrading, etc. mutant ATXN2 (or other protein including an elongatedpolyglutamine tract or similar expansion tract, or other mutant proteinassociated with neurological disorders, such as those described above)to minimize aggregation of Staufen1 thereto, stabilization of Staufen1,the like, or a combination thereof.

The same types of Staufen1-regulating agents described herein supra canalso be employed in the method of controlling Staufen1 accumulation in atarget cell. Additionally, the same methods of introducing theStaufen1-regulating agent to the target cell as described herein supracan likewise be used in the method of controlling Staufen1 accumulationin a target cell. Further, the same or similar amounts as describedherein supra may be employed in the method of controlling Staufen1accumulation in a target cell, depending on the particular patient, typeand severity of the condition, dosing regimen, Staufen1-regulating agentbeing employed, etc.

The method of treating a neurodegenerative condition associated withStaufen1-induced dysregulation of RNA metabolism can includeadministering a therapeutically effective amount of aStaufen1-regulating agent to a subject. It is noted that there are anumber of neurodegenerative conditions that can be treated viaadministration of a therapeutically effective amount of aStaufen1-regulating agent. Non-limiting examples can includespinocerebellar ataxia type 1, spinocerebellar ataxia type 2,spinocerebellar ataxia type 3 (Machado-Joseph disease), spinocerebellarataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxiatype 8, spinocerebellar ataxia type 17, Huntington's disease,amyotrophic lateral sclerosis, Alzheimer's disease, frontotemporaldementia, Fragile X syndrome, Mytonic dystrophy, the like, or acombination thereof.

The Staufen1-regulating agent can be administered in a number of ways.Non-limiting examples can include administration via injection (e.g.intravenous, intrathecal, etc.), oral/enteral administration,transmucosal administration, transdermal administration, implantation,or a combination thereof.

Depending on the mode of administration, the particularStaufen1-regulating agent(s) being employed, the type and severity ofthe condition, the amount of Staufen1-regulating agent beingadministered, etc., administration can be performed at a variety offrequencies. In some examples, it can be advantageous to administer theStaufen1-regulating agent one or more times per day (e.g. one, two,three, or four times per day). In other examples, it can be advantageousto administer the Staufen1-regulating agent from once every two days toonce every seven days, from once per week to once every two weeks, fromonce every two weeks to once every six weeks, from once every month toonce every 12 months, from once every six months to once every 18months, or a combination thereof.

The amount of Staufen1-regulating agent administered can likewise varydepending on the particular subject being treated, the particularStaufen1-regulating agent(s) being employed, the type and severity ofthe condition, mode of administration, etc. In some examples, thetherapeutically effective amount or dose can be an amount from about0.001 μg per kg of body weight to about 100 g per kg of body weight. Insome additional examples, the therapeutically effective amount or dosecan be an amount from about 0.001 μg per kg of body weight to about 0.1μg per kg of body weight. In other examples, the therapeuticallyeffective amount or dose can be from about 0.1 μg per kg of body weightto about 10 mg per kg of body weight. In still other examples, thetherapeutically effective amount or dose can be from about 1 mg per kgof body weight to about 50 mg per kg of body weight. In additionalexamples, the therapeutically effective amount or dose can be from about50 mg per kg body weight to about 500 mg per kg body weight. In furtherexamples, the therapeutically effective amount or dose can be from about500 mg per kg body weight to about 1000 mg per kg body weight. In stillfurther examples, the therapeutically effective amount or dose can befrom about 1000 mg per kg body weight to about 10 g per kg body weight.In additional examples, the therapeutically effective amount or dose canbe from about 10 g per kg body weight to about 100 g per kg body weight.In some specific examples, the therapeutically effective amount or dosecan be an amount from about 0.1 mg to about 1 mg, from about 1 mg toabout 15 mg, from about 15 mg to about 50 mg, from about 50 mg to about100 mg, or from about 100 mg to about 1000 mg. It is noted that theseamounts are based on the Staufen1-regulating agent itself and does notaccount for any ligands, delivery vectors, etc.

In some examples, where the Staufen1-regulating agent is administeredvia a viral vector, the therapeutically effective amount can be fromabout 1×10¹ to about 1×10²⁰ viral particles. In other examples, thetherapeutically effective amount can include from about 1×10⁵ to about1×10¹⁰ viral particles. In other examples, the therapeutically effectiveamount can include from about 1×10⁷ to about 1×10¹² viral particles. Inyet other examples, the therapeutically effective amount can includefrom about 1×10⁹ to about 1×10¹⁵ viral particles. In further examples,the therapeutically effective amount can include from about 1×10¹ toabout 1×10²⁰ viral particles.

The therapeutically effective amount of the Staufen1-regulating agentcan depend on the mode of administration, the subject being treated, thetype and severity of the condition, the particular Staufen1-regulatingagent(s) being employed, etc. In some examples, the therapeuticallyeffective amount of Staufen1-regulating agent can be an amountsufficient to increase PCP2 mRNA levels in the target cell, CALB1 mRNAlevels in the target cell, other mRNAs in the target cell that aremetabolized via Staufen1-associated metabolism, or a combination thereofas compared to the levels of these mRNAs prior to or without introducingthe Staufen1-regulating agent. In some examples, the therapeuticallyeffective amount can be an amount sufficient to increase one or more ofthese mRNAs by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, ormore as compared to the level prior to or without administration of theStaufen1-regulating agent. In some examples, the therapeuticallyeffective amount of the Staufen1-regulating agent can be an amountsufficient to reduce the amount of Staufen1 present in the target cellas compared to the amount of Staufen1 present in the target cell priorto or without administration of the Staufen1-regulating agent. In somespecific examples, the therapeutically effective amount can be an amountsufficient to reduce Staufen1 in the target cell by at least 30%, 40%,50%, 60%, or 70% as compared to Staufen1 levels prior to or withoutadministration of the Staufen1-regulating agent.

In still other examples, the therapeutically effective amount of theStaufen1-regulating agent can be an amount sufficient to reduce a mutantATXN2 level (or a level of another protein including an elongatedpolyglutamine tract or similar expansion tract, or other mutant proteinassociated with neurological disorders, such as those described above)in the target cell as compared to the mutant ATXN2 level (or mutantc9orf72 level, or mutant TDP-43 level, or mutant Tau level, or mutantAPP level, for example) prior to or without administration of theStaufen1-regulating agent. In some specific examples, thetherapeutically effective amount can be an amount sufficient to reducemutant ATXN2 (or other protein including an elongated polyglutaminetract or similar expansion tract, or other mutant protein associatedwith neurological disorders, such as those described above) in thetarget cell by at least 30%, 40%, 50%, 60%, or 70% as compared to mutantATXN2 levels (or mutant c9orf72 levels, or mutant TDP-43 levels, ormutant Tau levels, or mutant APP levels, for example) prior to orwithout administration of the Staufen1-regulating agent (e.g. where thestaufen1-regulating agent is a mutant ATXN2-inhibiting agent, a mutantc9orf72-inhibiting agent, a mutant TDP-43-inhibiting agent, a mutantTau-inhibiting agent, and/or a mutant APP-inhibiting agent, forexample). Additionally, in some further examples, the therapeuticallyeffective amount can be an amount sufficient to minimize furtherdepletion of motor performance of the subject, restore a portion ofpreviously depleted motor performance, or both.

A therapeutic agent, such as a Staufen1-regulating agent for treating aneurodegenerative condition associated with Staufen1-induceddysregulation of RNA metabolism can include those described elsewhereherein. A therapeutic composition for treating a neurodegenerativecondition associated with Staufen1-induced dysregulation of RNAmetabolism can include a therapeutically effective amount ofStaufen1-regulating agent and a pharmaceutically acceptable carrier.Again, the Staufen1-regulating agent in the therapeutic composition caninclude those described elsewhere herein.

The therapeutically effective amount of the Staufen1-regulating agentcan depend on the mode of administration, the subject being treated, thetype and severity of the condition, the particular Staufen1-regulatingagent(s) being employed, etc. In some examples, the therapeuticallyeffective amount of Staufen1-regulating agent can be an amountsufficient to increase PCP2 mRNA levels in the target cell, CALB1 mRNAlevels in the target cell, other mRNAs in the target cell that aremetabolized via Staufen1-associated metabolism, or a combination thereofwhen administered in an effective dosing regimen as compared to thelevels of these mRNAs prior to or without administration of theStaufen1-regulating agent. In some examples, the therapeuticallyeffective amount can be an amount sufficient to increase one or more ofthese mRNAs by at least 10%, 20%, 30%, 40%, or more when administered inan effective dosing regimen as compared to the level prior to or withoutadministration of the Staufen1-regulating agent. In some examples, thetherapeutically effective amount of the Staufen1-regulating agent can bean amount sufficient to reduce the amount of Staufen1 present in thetarget cell when administered in an effective dosing regimen as comparedto the amount of Staufen1 present in the target cell prior to or withoutadministration of the Staufen1-regulating agent. In some specificexamples, the therapeutically effective amount can be an amountsufficient to reduce Staufen1 in the target cell by at least 30%, 40%,50%, 60%, or 70% when administered in an effective dosing regimen ascompared to Staufen1 levels prior to or without administration of theStaufen1-regulating agent.

In still other examples, the therapeutically effective amount of theStaufen1-regulating agent can be an amount sufficient to reduce a mutantATXN2 level (or a level of another protein including an elongatedpolyglutamine tract or similar expansion tract, or other mutant proteinassociated with neurological disorders, such as those described above)in the target cell when administered in an effective dosing regimen ascompared to the mutant ATXN2 level (or mutant c9orf72 level, or mutantTDP-43 level, or mutant Tau level, or mutant APP level, for example)prior to or without administration of the Staufen1-regulating agent. Insome specific examples, the therapeutically effective amount can be anamount sufficient to reduce ATXN2 (or other protein including anelongated polyglutamine tract or similar expansion tract, or othermutant protein associated with neurological disorders, such as thosedescribed above) in the target cell by at least 30%, 40%, 50%, 60%, or70% when administered in an effective dosing regimen as compared toATXN2 levels (or mutant c9orf72 levels, or mutant TDP-43 levels, ormutant Tau levels, or mutant APP levels, for example) prior to orwithout administration of the Staufen1-regulating agent.

In some specific examples, the therapeutically effective amount can bean amount from about 1 picomolar (pM) to about 100 millimolar (mM). Insome other examples, the therapeutically effective amount can be anamount from about 1 μM to about 100 μM. In still other examples, thetherapeutically effective amount can be an amount from about 50 μM toabout 500 μM. In yet other examples, the therapeutically effectiveamount can be an amount from about 300 μM to about 1000 μM. Inadditional examples, the therapeutically effective amount can be anamount from about 1 nanomolar (nM) to about 50 nM. In still additionalexamples, the therapeutically effective amount can be from about 10 nMto about 500 nM. In further examples, the therapeutically effectiveamount can be an amount from about 400 nM to about 1 mM. In stillfurther examples, the therapeutically effective amount can be an amountfrom about 700 nM to about 10 mM.

In some other specific examples, the therapeutically effective amountcan be from about 0.001 μg of Staufen1-regulating agent per gram (g) oftherapeutic composition (μg/g) to about 200 mg of Staufen1-regulatingagent per gram of therapeutic composition (mg/g). In other examples, thetherapeutically effective amount can be from about 0.01 μg/g to about 1μg/g Staufen1-regulating agent in the therapeutic composition. In someother examples, the therapeutically effective amount can be from about0.1 μg/g to about 10 μg/g Staufen1-regulating agent in the therapeuticcomposition. In yet other examples, the therapeutically effective amountcan be from about 1 μg/g to about 100 μg/g Staufen1-regulating agent inthe therapeutic composition. In still other examples, thetherapeutically effective amount can be from about 10 μg/g to about 1mg/g Staufen1-regulating agent in the therapeutic composition. In someadditional examples, the therapeutically effective amount can be fromabout 100 μg/g to about 10 mg/g Staufen1-regulating agent in thetherapeutic composition. In some further examples, the therapeuticallyeffective amount can be from about 1 mg/g to about 100 mg/gStaufen1-regulating agent in the therapeutic composition. It is againnoted that the therapeutically effective amounts disclosed herein aregenerally based on the amount of Staufen1-regulating agent itselfwithout any associated ligands, delivery vehicles, etc., unlessotherwise specified.

In some additional specific examples, where the Staufen1-regulatingagent is carried by a viral vector, the therapeutically effective amountcan include from about 1×10⁵ to about 1×10¹⁵ viral particles per gram oftherapeutic composition. In some examples, the therapeutically effectiveamount can include from about 1×10⁵ to about 1×10⁷ viral particles pergram of therapeutic composition. In some additional examples, thetherapeutically effective amount can include from about 1×10⁷ to about1×10⁹ viral particles per gram of therapeutic composition. In some otherexamples, the therapeutically effective amount can include from about1×10⁹ to about 1×10¹¹ viral particles per gram of therapeuticcomposition. In yet other examples, the therapeutically effective amountcan include from about 1×10¹¹ to about 1×10¹³ viral particles per gramof therapeutic composition. In still other examples, the therapeuticallyeffective amount can include from about 1×10¹³ to about 1×10¹⁵ viralparticles per gram of therapeutic composition.

The pharmaceutically acceptable carrier can be formulated for a varietyof modes of administration. For example, the pharmaceutically acceptablecarrier can be formulated to administer the Staufen1-regulating agentvia injection (e.g. intravenous, intrathecal, etc.), oral/enteraladministration, transdermal administration, transmucosal administration,inhalation, implantation, or the like.

In some examples, the pharmaceutically acceptable carrier can beformulated to provide a therapeutic composition for administration viainjection, such as intramuscular injection, intravenous injection,subcutaneous injection, intradermal injection, intrathecal injection, orthe like. In such examples, the pharmaceutically acceptable carrier caninclude a variety of components, such as water, a solubilizing agent, adispersing agent, a tonicity agent, a pH adjuster, a buffering agent, apreservative, a chelating agent, a bulking agent, the like, or acombination thereof.

In some examples, an injectable therapeutic composition can include asolubilizing or dispersing agent. Non-limiting examples of solubilizingor dispersing agents can include polyoxyethylene sorbitan monooleates,lecithin, polyoxyethylene polyoxypropylene co-polymers, propyleneglycol, glycerin, ethanol, polyethylene glycols, sorbitol,dimethylacetamide, polyethoxylated castor oils, n-lactamide,cyclodextrins, caboxymethyl cellulose, acacia, gelatin, methylcellulose, polyvinyl pyrrolidone, the like, or combinations thereof.

In some examples, an injectable therapeutic composition can include atonicity agent. Non-limiting examples of tonicity agents can includesodium chloride, potassium chloride, calcium chloride, magnesiumchloride, mannitol, sorbitol, dextrose, glycerin, propylene glycol,ethanol, trehalose, phosphate-buffered saline (PBS), Dulbecco's PBS,Alsever's solution, Tris-buffered saline (TBS), water, balanced saltsolutions (BSS), such as Hank's BSS, Earle's BSS, Grey's BSS, Puck'sBSS, Simm's BSS, Tyrode's BSS, and BSS Plus, the like, or combinationsthereof. The tonicity agent can be used to provide an appropriatetonicity of the therapeutic composition. In one aspect, the tonicity ofthe therapeutic composition can be from about 250 to about 350milliosmoles/liter (mOsm/L). In another aspect, the tonicity of thetherapeutic composition can be from about 277 to about 310 mOsm/L.

In some examples, an injectable therapeutic composition can include a pHadjuster or buffering agent. Non-limiting examples of pH adjusters orbuffering agents can include a number of acids, bases, and combinationsthereof, such as hydrochloric acid, phosphoric acid, citric acid, sodiumhydroxide, potassium hydroxide, calcium hydroxide, acetate buffers,citrate buffers, tartrate buffers, phosphate buffers, triethanolamine(TRIS) buffers, the like, or combinations thereof. Typically, the pH ofthe therapeutic composition can be from about 5 to about 9, or fromabout 6 to about 8. However, other suitable pHs can also be desirable.

In some examples, an injectable therapeutic composition can include apreservative. Non-limiting examples of preservatives can includeascorbic acid, acetylcysteine, bisulfite, metabisulfite,monothioglycerol, phenol, meta-cresol, benzyl alcohol, methyl paraben,propyl paraben, butyl paraben, benzalkonium chloride, benzethoniumchloride, butylated hydroxyl toluene, myristyl gamma-picolimiumchloride, 2-phenoxyethanol, phenyl mercuric nitrate, chlorobutanol,thimerosal, tocopherols, the like, or combinations thereof.

In some examples, an injectable therapeutic composition can include achelating agent. Non-limiting examples of chelating agents can includeethylenediaminetetra acetic acid, calcium, calcium disodium,versetamide, calteridol, diethylenetriaminepenta acetic acid, the like,or combinations thereof.

In some examples, an injectable therapeutic composition can include abulking agent. Non-limiting examples of bulking agents can includesucrose, lactose, trehalose, mannitol, sorbitol, glucose, rafinose,glycine, histidine, polyvinyl pyrrolidone, the like, or combinationsthereof.

In yet other examples, the pharmaceutically acceptable carrier can beformulated to provide a therapeutic composition for enteraladministration, such as via solid oral dosage forms or liquid oraldosage forms. In the case of solid oral dosage forms, thepharmaceutically acceptable carrier can include a variety of componentssuitable for forming a capsule, tablet, or the like. In the case of aliquid dosage form, the pharmaceutically acceptable carrier can includea variety of components suitable for forming a dispersion, a suspension,a syrup, an elixir, or the like.

In some specific examples, the therapeutic composition can be formulatedas a tablet. In such examples, the therapeutic composition can typicallyinclude a binder. Non-limiting examples of binders can include lactose,calcium phosphate, sucrose, corn starch, microcrystalline cellulose,gelatin, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),hydroxypropyl cellulose, hydroxyethylcellulose, carboxymethyl cellulose(CMC), the like, or combinations thereof.

Where the therapeutic composition is formulated as a tablet, in someexamples the therapeutic composition can also include a disintegrant.Non-limiting examples of disintegrants can include crosslinked PVP,crosslinked CMC, modified starch, sodium starch glycolate, the like, orcombinations thereof.

In some examples, the tablet can also include a filler. Non-limitingexamples of fillers can include lactose, dicalcium phosphate, sucrose,microcrystalline cellulose, the like, or combinations thereof.

In some further examples, the tablet can include a coating. Suchcoatings can be formed with a variety of materials, such ashydroxypropyl methylcellulose (HPMC), shellac, zein, variouspolysaccharides, various enterics, the like, or combinations thereof.

In some examples, the tablet can include a variety of other ingredients,such as anti-adherents (e.g. magnesium stearate, for example),colorants, glidants (e.g. fumed silica, talc, magnesium carbonate, forexample), lubricants (e.g. talc, silica, magnesium stearate, stearicacid, for example) preservatives, desiccants, and/or other suitabletablet excipients, as desired.

In some other examples, the therapeutic composition can be formulated asa capsule. In such examples, the capsule itself can typically includegelatin, hypromellose, HPMC, CMC, the like, or combinations thereof. Avariety of excipients can also be included within the capsule, such asbinders, disintegrants, fillers, glidants, preservatives, coatings, thelike, or combinations thereof, such as those listed above with respectto tablets, for example, or other suitable variations.

In some examples, the therapeutic composition can be formulated as aliquid oral dosage form. A liquid oral dosage form can include a varietyof excipients, such as a liquid vehicle, a solubilizing agent, athickener or dispersant, a preservative, a tonicity agent, a pH adjusteror buffering agent, a sweetener, the like, or a combination thereof.Non-limiting examples of liquid vehicles can include water, ethanol,glycerol, propylene glycol, the like, or combinations thereof.Non-limiting examples of solubilizing agents can include banzalkoniumchloride, benzethonium chloride, cetylpyridinium chloride, docusatesodium, nonoxynol-9, octoxynol, polyoxyethylene polyoxypropyleneco-polymers, polyoxyl castor oils, polyoxyl hydrogenated castor oils,polyoxyl oleyl ethers, polyoxyl cetylstearyl ethers, polyoxyl stearates,polysorbates, sodium lauryl sulfate, sorbitan monolaurate, sorbitanmonooleate, sorbitan monopalmitate, sorbitan monostearate, tyloxapol,the like, or combinations thereof. Non-limiting examples of thickenersor dispersants can include sodium alginate, methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose, HPMC, CMC,microcrystalline cellulose, tragacanth, xanthangum, bentonite,carrageenan, guar gum, colloidal silicon dioxide, the like, orcombinations thereof. The preservative, tonicity agent, pH adjuster orbuffering agent can typically be any of those described above withrespect to the injectable formulations or other suitable preservative,tonicity agent, pH adjuster or buffering agent. Sweeteners can includenatural and/or artificial sweeteners, such as sucrose, glucose,fructose, stevia, erythritol, xylitol, aspartame, sucralose, neotame,acesulfame potassium, saccharin, advantame, sorbitol, the like, orcombinations thereof, for example.

In yet other examples, the pharmaceutically acceptable carrier can beformulated to provide a therapeutic composition for transdermal ortransmucosal administration, such as via the skin, the nasal cavity, thelike, or a combination thereof. Where the therapeutic composition isformulated for transdermal or transmucosal administration, thepharmaceutically acceptable carrier can include a variety of componentssuitable for forming a suspension, dispersion, lotion, cream, ointment,gel, foam, patch, powder, paste, sponge, the like, or a combinationthereof. Non-limiting examples can include a solubilizer, an emulsifier,a dispersant, a thickener, an emollient, a pH adjuster, a tonicityagent, a preservative, an adhesive, a penetration enhancer, the like, ora combination thereof. Non-limiting examples of solubilizers and/oremulsifiers can include water, ethanol, propylene glycol, ethyleneglycol, glycerin, polyethylene glycol, banzalkonium chloride,benzethonium chloride, cetylpyridinium chloride, docusate sodium,nonoxynol-9, octoxynol, polyoxyethylene polyoxypropylene co-polymers,polyoxyl castor oils, polyoxyl hydrogenated castor oils, polyoxyl oleylethers, polyoxyl cetylstearyl ethers, polyoxyl stearates, polysorbates,sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate,sorbitan monopalmitate, sorbitan monostearate, tyloxapol, the like, orcombinations thereof. In some examples, the solubilizer can also includea hydrocarbon or fatty substance, such as petrolatum, microcrystallinewax, paraffin wax, mineral oil, ceresi, coconut oil, bees wax, oliveoil, lanolin, peanut oil, spermaceti wax, sesame oil, almond oil,hydrogenated castor oils, cotton seed oil, soybean oil, corn oil,hydrogenated sulfated castor oils, cetyl alcohol, stearyl alcohol, oleylalcohol, lauryl alcohol, myristyl alcohol, stearic acid, oleic acid,palmitic acid, lauraic acid, ethyl oleate, isopropyl myristicate, thelike, or combinations thereof. In some examples, the solubilizer caninclude a silicon, such as polydimethylsiloxanes, methicones,dimethylpropylsiloxanes, methyl phenyl polysiloxanes, steryl esters ofdimethyl polysiloxanes, ethoxylated dimethicones, ethoxylatedmethicones, the like, or combinations thereof.

In some additional examples, the therapeutic composition can include adispersant and/or thickening agent, such as polyacrylic acids (e.g.Carbopols, for example), gelatin, pectin, tragacanth, methyl cellulose,hydroxylethylcellulose, hydroxypropylcellulose, HPMC, CMC, alginate,starch, polyvinyl alcohol, polyvinyl pyrrolidone, co-polymers ofpolyoxyethylene and polyoxypropylene, polyethylene glycol, the like, orcombinations thereof.

In some examples, the therapeutic composition can include an emollient,such as aloe vera, lanolin, urea, petrolatum, shea butter, cocoa butter,mineral oil, paraffin, beeswax, squalene, jojoba oil, coconut oil,sesame oil, almond oil, cetyl alcohol, stearyl alcohol, olive oil, oleicacid, triethylhexanoin, glycerol, sorbitol, propylene glycol,cyclomethicone, dimethicone, the like, or combinations thereof.

In some examples, the therapeutic composition can include an adhesive,such as acrylic adhesives, polyisobutylene adhesives, silicon adhesives,hydrogel adhesives, the like, or combinations thereof.

In some examples, the therapeutic composition can include a penetrationenhancer, such as ethanol, propylene glycol, oleic acid and other fattyacids, azone, terpenes, terpenoids, bile acids, isopropyl myristate andother fatty esters, dimethyl sulphoxides, N-methyl-2-pyrrolidone andother pyrrolidones, the like, or combinations thereof.

The pH adjusters, tonicity agents, and preservatives in the topical,transdermal, or transmucosal therapeutic composition can generallyinclude those pH adjusters and buffering agents, tonicity agents, andpreservative agents listed above, or any other suitable pH adjusters,buffering agent, tonicity agent, or preservative for a particularformulation and/or use thereof. In some examples, the therapeuticcomposition can also include fumed silica, mica, talc, titanium dioxide,kaolin, aluminum glycinate, ethylenediaminetetraacetic acid, fragrances,colorants, other components as described above, the like, orcombinations thereof.

In some additional examples, the pharmaceutically acceptable carrier canbe formulated for administration via inhalation. In some examples, suchformulations can include a propellant, such as hydrofluoralkanes, suchas HFA134a, HFA227, or other suitable propellant. In yet other examples,the therapeutic composition can be formulated for administration vianebulization. In either case, the therapeutic composition can alsoinclude a variety of solubilizing agents, such as those described above.In other examples, the therapeutic composition can be formulated as adry powder aerosol. In some examples, the therapeutic composition caninclude a particulate carrier and/or other particulate excipients, suchas lactose, mannitol, other crystalline sugars, fumed silica, magnesiumstearate, amino acids, the like, or combinations thereof.

In still additional examples, the pharmaceutically acceptable carriercan be formulated as a biodegradable matrix for implantation into asubject. Specific non-limiting examples of suitable matrix materials caninclude biodegradable polymers such as PLGA (different ratios of lacticto glycolide content and end groups such as acid or ester termination),PVA, PEG, PLA, PGA, HPMC, hydroxypropylcellulose, sodiumcarboxymethylcellulose, croscarmellose sodium, poly caprolactone,hyaluronic acid, albumin, sodium chloride block copolymers thereof, andthe like. Specific copolymers such as polylactic-polyglycolic acid blockcopolymers (PLGA), polyglycolic acid-polyvinyl alcohol block copolymers(PGA/PVA), hydroxypropylmethylcellulose (HPMC),polycaprolactone-polyethylene glycol block copolymers, croscarmellose,and the like can be particularly effective. In one aspect, the activeagent matrix can be a PLGA having about 45-80% PLA and 55-20% PGA suchas about 65% PLA and 35% PGA.

In some examples, the biodegradable matrix can be configured tobiodegrade over a period of from about 1 week to about 2 weeks. In yetother examples, the biodegradable matrix can be configured to biodegradeover a period of from about 2 weeks to about 4 weeks. In still otherexamples, the biodegradable matrix can be configured to biodegrade overa period of from about 4 weeks to about 6 weeks or longer.

In some examples, the therapeutic composition can include a therapeuticagent (e.g. a supplementary therapeutic agent) in addition to theStaufen1-regulating agent. Non-limiting examples can include adopaminergic agent, a cholinesterase inhibitor, an antipsychotic agent,an analgesic, an anti-inflammatory agent, an inducer of autophagy (e.g.sirolimus, everolimus, tacrolimus, etc.), the like, or a combinationthereof.

The present disclosure also includes a method of treating a neurologicaldisorder or condition in a subject. The method can include identifying alevel of Staufen1 in a biological fluid, determining whether the levelof Staufen1 in the biological fluid indicates that the subject has or islikely to have a neurological disorder or condition, and administering aStaufen1-regulating agent to a subject who has, or is likely to have, aneurological disorder or condition. Biological fluids can includecerebral spinal fluid, blood serum, plasma, the like, or a combinationthereof.

Thus, Staufen1 can be used as an antigen or biomarker in a biologicalfluid to detect the presence or likelihood of a neurological disorder orcondition in a subject for subsequent treatment with aStaufen1-regulating agent. Further, because Staufen1 can be highlyelevated in individuals having a variety of neurological disorders orconditions, Staufen1 can be readily detectable, have a broad dynamicrange, and can be a marker for multiple disorders or conditions.

A variety of analytical techniques can be used to identify thepresence/absence of Staufen1 and/or quantify Staufen1 levels in abiological fluid. Some non-limiting examples can include liquidchromatography-mass spectrometry (LC/MS), liquid chromatography-tandemmass spectrometry (LC/MS/MS), an enzyme-linked immunosorbent assay(ELISA), an enzyme immunoassay (EIA), a meso scale discovery (MSD)assay, the like, or a combination thereof.

The present disclosure also describes a kit for detecting Staufen1 in abiological fluid. In some examples, the kit can be employed one or moreof the methods of treating a neurological disorder or condition in asubject as described above. In some examples, the kit can include aStaufen1 capture antibody, a Staufen1 detection antibody, or acombination thereof. In some further examples, one or more of theStaufen1 capture antibody and the Staufen1 detection antibody can belabeled. In some examples, the kit can include a Staufen1 captureantibody. In some examples, the kit can include a Staufen1 detectionantibody. In some examples, the kit can include both a Staufen1 captureantibody and a Staufen1 detection antibody. In some further examples,the kit can further include one or more suitable reagents, buffers, thelike, or a combination thereof to facilitate detection of Staufen1 inthe biological sample.

In still further examples, the kit can include instructions about how todetect Staufen1 in a biological sample. The instructions can vary,depending on the particular type of analytical methodology employed,instrumentation employed, etc.

EXAMPLES Example 1—Co-Localization of ATXN2 and Staufen1 in SCA2Fibroblasts and their Physical Interaction

An association of Staufen1 and SGs in brain oligodendrocytes and othercultured cells has been described. Because ATXN2 is a component of SGs,it was investigated whether ATXN2 and Staufen1 co-localized under stresscondition. HEK-293 cells were stressed by heat-shock and the cellularlocalization of ATXN2 and Staufen1 were studied. Under physiologiccondition, ATXN2 and Staufen immunostaining demonstrated that both ATXN2and Staufen1 are distributed throughout the cytoplasm in HEK-293 cells(FIG. 1A). However, when cells were exposed to heat-shock,co-localization of ATXN2 and Staufen1 was observed in distinctcytoplasmic foci resembling SGs (FIG. 1A). Additional co-localizationstudies were performed using TIA-1 antibodies, a protein marker for SGs.ATXN2 and TIA-1 did co-localize in SGs in cells upon heat-shocktreatment (FIG. 1A). Thus, both ATXN2 and Staufen1 were co-localized toSGs under stress conditions (heat-shock).

As ATXN2 aggregations are evident in SCA2 brain, the recruitment ofStaufen1 in SCA2 fibroblasts under normal and stress conditions wasinvestigated. When normal (ATXN2-Q22/22) and SCA2 fibroblasts(ATXN2-Q22/42) were stained with ATXN2 and Staufen1 antibodies,stress-granule-like aggregates were seen in SCA2 fibroblasts with theco-localization of mutant ATXN2 and Staufen1 but not in normalfibroblasts. By ImageJ, the average area per aggregate was found to beabout 15% higher for SCA2 cells over wild type cells (FIGS. 1B-1C).Interestingly, increased number of granules co-localizing ATXN2 andStaufen1 were seen in normal and SCA2 fibroblasts under stressconditions but granule numbers were more pronounced in SCA2 cells (FIGS.1D and 1E).

Co-localization of ATXN2 and Staufen1 in SG-like aggregates in SCA2cells predicts physical interaction between these two proteins in vivo.To test this idea, protein immunoprecipitation (IP) experiments wereperformed in cultured HEK-293 cells overexpressing Flag-tagged ATXN2containing -Q22 or -Q108 repeats. Whole cell extracts (with or withoutRNase A treatment) were subjected to immunoprecipitation with Flag mAbbeads and eluted bound protein complexes were analyzed by western blot.Western blot analyses of the eluted proteins showed expression ofFlag-ATXN2 proteins and RNA-dependent interaction of endogenous Staufen1with Flag-ATXN2-Q22 or Flag-ATXN2-Q108 (FIG. 1F). The immunoprecipitatesalso showed co-IP of PABPC1 (RNA-dependent) and DDX6 (both RNA- andnon-RNA-dependent), which can interact with ATXN2 in vivo. Furthermore,the IP products were analyzed by tandem mass spectrometry and confirmedthe authenticity of interaction between ATXN2 and Staufen1. Togetherthese data indicate the physical interaction and co-localization ofATXN2 and Staufen1 in stress-granule-like aggregates in SCA2 cells.

Example 2—Staufen1 Expression in Mouse Cerebellum

As cerebellar degeneration is prominent in SCA2, the expression ofStaufen1 in cerebellar sections in wild-type mice was examined.Immunostaining with anti-Staufen antibody revealed expression ofStaufen1 in cerebellum (FIG. 2A). Consistent with this finding, theHuman Protein Atlas shows widespread expression of Staufen1 withsignificant expression level in the cerebellum. The specificity ofanti-Staufen antibody was confirmed by measuring endogenous Staufen1levels in western blot analyses using cell extracts of human HEK-293cells or mouse N2a neuroblastoma cells treated with siRNAs directedagainst human or mouse Staufen1 (FIGS. 2B and 2C).

Example 3—Steady-State Staufen1 Levels are Increased in SCA2 PatientCell Lines and BAC-SCA2 Mouse Cerebella

Because Staufen1 interacts with ATXN2 and forms aggregates with mutantATXN2 in SCA2 fibroblasts, steady-state levels of Staufen1 inSCA2-fibroblasts and -lymphoblastoid B (LB) cells were measured. Thecells used were (a) four normal skin fibroblasts with Q22 repeats, (b)five SCA2 skin fibroblasts with Q35, Q35, Q35, Q42 and Q45 repeats, (c)two normal LB cells with Q22 repeats, and (d) three SCA2-LB cells withQ40, Q46, Q52 repeats in the ATXN2 gene. Whole cell extracts wereprepared from harvested cells by using Laemmli SDS-PAGE sample bufferand analyzed by western blot to measure Staufen1 steady-state levels.Steady-state levels of 55-kD Staufen1 isoform were ˜4.0, ˜4.8, ˜4.5,˜3.6 and ˜4.9 fold elevated in five SCA2 fibroblasts when compared withfour normal fibroblasts. Similarly, three SCA2-LB cells alsodemonstrated ˜3.7, ˜3.2 and ˜4.1 fold increased level of Staufen1 whencompared with two normal LB cells (FIGS. 3A-3B). The level of DDX6protein, another ATXN2 interactor, was not significantly altered inSCA2-fibroblasts or -LB cells when compared with normal cells. Todetermine whether Staufen1 levels were affected specifically by themutant ATXN2 in vivo, the relative levels of Staufen1 were measured bywestern blotting in cerebellar extracts from BAC-SCA2 mouse models at 24weeks of age. An increase in abundance of Staufen1 in BAC-Q72 mice(˜1.81 and ˜2.01 fold) was observed, but the levels were unaltered inBAC-Q22 mice when compared with wild-type mice (FIG. 3C). Conversely, nosignificant alteration of Staufen1 levels was observed in SCA3 patientfibroblasts or HEK-293 cells expressing mutant ATXN3 (Flag-ATXN3-Q56)when compared with control (FIGS. 3A-3B and 3D). Together, thesefindings established that the level of Staufen1 was elevated in SCA2disease. The Staufen1 elevation was consistent in the multiple celllines and in vivo.

Next, steady-state Staufen1 at protein and mRNA levels were measured inHEK-293 cells in which ATXN2 levels were reduced by siATXN2 andcerebella from Atxn2 knock-out mice (8 weeks of age) by qPCR. In theseexperiments, Staufen1 steady-state protein and mRNA levels were notaltered in HEK-293 cells with reduced ATXN2 levels or in Atxn2 knock-outmice (FIGS. 3E-3G). Thus, increased abundance of Staufen1 in SCA2 didnot result from the functional loss of ataxin-2.

Example 4—Expression of Mutant ATXN2 in HEK-293 Cells RecapitulatesIncreased Abundance of Staufen1 and Stability of Staufen1 Protein isIncreased in SCA2fibroblasts

To test whether increased Staufen1 levels could be recapitulated uponexpression of ATXN2 with long polyQ tracts, steady-state levels ofStaufen1 were measured in whole cell extracts of HEK-293 cellsexpressing Flag-tagged ATXN2-Q22 or -Q58 or -Q108. Western blot analysesindicated that expression of ATXN2-Q58 or -Q108 was sufficient to resultin increased Staufen1 levels (˜2.2 and ˜2.7 fold) when compared withATXN2-Q22 and control (FIG. 4A). To asses if the increased Staufen1level was not a consequence of selective cellular toxicity of ATXN2-Q58or -Q108 expression, endogenous PABPC1 and DDX6 levels were measured inthose cell extracts by western blot. The levels of PABPC1 and DDX6 werenot altered in HEK-293 cells expressing ATXN2-Q22 or -Q58 or -Q108 (FIG.4A). Thus, these data demonstrate that the increased abundance ofStaufen1 occurs specifically as a consequence of mutant ATXN2 expressionin HEK-293 cells.

To identify the underlying mechanism of ATXN2 polyQ expansion-mediatedStaufen1 abundance, Staufen1 transcript levels were examined in normaland SCA2 fibroblasts. Results of qPCR data showed little or nodifferences in Staufen1 transcript levels (data not shown) and suggestedthat ATXN2 polyQ expansion likely affected Staufen1 protein levels. Totest this, comparative protein stability for Staufen1 was measured bycycloheximide (CHX)-chase assay in normal and SCA2 fibroblasts. RelativeStaufen1 levels were analyzed from harvested normal and SCA2 fibroblastextracts. (FIGS. 4B and 4C). Western blot analyses demonstrated thatStaufen1 levels were reduced 4 hr after CHX treatment in normalfibroblasts. However, in SCA2 fibroblasts, Staufen1 protein expressionlevels showed relatively little decrease. p21 Waf1/Cip1 levels were alsomeasured as experimental control and demonstrated that p21 Waf1/Cip1levels were reduced by ˜90% at 4 hr after CHX treatment in both normaland SCA2 fibroblasts. Thus, these data suggest that presence of mutantATXN2 in SCA2 cells increases the stability of Staufen1 protein.

Example 5—Elevated Level of Staufen1 Results in Aberrant Processing ofRNA Targets

To investigate the role of Staufen1 in the development of the aberrantgene expression patterns observed in SCA2, the effect of alteredStaufen1 dosage on PCP2 and CALB1 gene expression in HEK-293 cells wasevaluated and these results were compared with those observed in SCA2-LBcells. First, PCP2 mRNA levels were measured in SCA2-LB (ATXN2-Q22/52)cells using qPCR. CALB1 mRNA could not be analyzed due to undetectableexpression levels. The levels of PCP2 mRNA were reduced by 45.4±7.7% inSCA2-LB cells when compared with normal LB (ATXN2-Q22/22) cells (FIG.5A). In contrast, no alteration of ATXN2 transcript levels was observedin normal and SCA2-LB cells. In separate experiments, the role ofincreased Staufen1 levels on PCP2 and CALB1 mRNA abundance in HEK-293cells were evaluated. Staufen1 protein was overexpressed in a range thatmimicked the changes that are observed in SCA2-fibroblasts or -LB cells.Elevated expression of Staufen1 induced the reduction of PCP2 and CALB1mRNA levels by 40.8±7.9% and 32.8±8.5%, respectively. To excludeselective cellular toxicity of Staufen1 overexpression, endogenous ATXN2mRNA levels were measured. The levels of ATXN2 transcript was notaltered in HEK-293 cells overexpressing Staufen1 (FIGS. 5B and 5C).Thus, these data demonstrate that aberrant gene expression patterns inSCA2 cells can be recapitulated in cell culture model.

Next, PCP2 protein abundance upon increased Staufen1 levels wasanalyzed. Endogenous PCP2 protein could not be analyzed (due toundetectable expression levels), but exogenous MYC-tagged PCP2 wasexpressed in short-term hygromycin selected HEK-293 cells expressingFlag-tagged Staufen1 and tested regulatory effects of increased Staufen1levels on exogenous MYC-tagged PCP2 expression. MYC-tagged PCP2 cDNAincluding 5′ and 3′ UTRs (i) or excluding 3′UTR (ii) were cloned underthe transcriptional control of the CMV promoter. Forty-eight hourspost-transfection, western blot analyses revealed that the exogenousPCP2 levels were significantly reduced in Staufen1 expressing cellstransfected with MYC-tagged PCP2 cDNA[(5′+3′)UTR] (i) when compared withcontrol. However, increased Staufen1 levels did not show inhibitoryeffect on expression of MYC-tagged PCP2 cDNA(+5′UTR) (ii) compared withcontrol (FIGS. 5D-5F). To control for equal transfection, Neomycinprotein levels were monitored, which was expressed as an independentcassette in MYC-tagged PCP2 plasmids. Thus, these data suggest thatStaufen1 regulates the expression of PCP2 via its 3′UTR.

Example 6—Staufen1 Interacts with PCP2 and CALB1 RNAs

Reduction of PCP2 and CALB1 mRNAs, and a concomitant repression of PCP2expression at protein levels by the elevated Staufen1 level predict thatStaufen1 might interact with subset of mRNAs and regulate theirexpressions. To identify Staufen1 targets, protein-RNAimmunoprecipitation (IP) experiments were performed in cultured HEK-293cells overexpressing Flag-tagged Staufen1. Non-RNase A treated wholecell extracts were subjected to immunoprecipitation with Flag mAb beadsand eluted protein-RNA complexes were divided as two aliquots forwestern blot and RT-PCR analyses. Flag-Staufen1 showed co-IP of ATXN2(FIG. 6A). RT-PCR analyses from the second aliquot revealed thatFlag-Staufen1 pulled down PCP2 and CALB1 mRNAs but not control GAPDHmRNA (FIG. 6A).

To confirm direct binding between Staufen1 and its targets, northwesternblotting experiments were performed using bacterially expressedrecombinant His-tagged Staufen1 protein and in vitro transcribedDIG-labelled PCP2 RNA probes. Several probes were generated that allowedtesting of which parts of PCP2 RNA interacted with Staufen1. The probesused were: DIG-PCP2[(5′+3′)UTR)] (i), DIG-PCP2(3′UTR) (ii), andDIG-PCP2(5′UTR) (iii) (FIG. 6B). Staufen1 showed binding towardPCP2[(5′+3′)UTR)] (i) or PCP2(3′UTR) (ii) RNAs but not to PCP2(5′UTR)(iii) RNA, thereby showing the specificity of the interaction (FIG. 6C).Together, these results demonstrate that both in vivo and in vitro,Staufen1 interacts with PCP2 mRNA through 3′UTR.

Example 7—Silencing of Staufen or Mutant ATXN2 Restores PCP2 mRNA Levelsin SCA2 Cells

The siRNAs used in this study were: All Star Negative Control siRNA(Qiagen Inc., USA; Cat #1027280), siATXN2 [Hs_ATXN2_2, (Qiagen Inc.,USA; Cat #SI00308196), human siSTAU: 5′ CCUAUAACUACAACAUGAGdTdT-3′ andmouse siStau1: 5′-CAACUGUACUACCUUUCC AdTdT-3′. Staufen1 siRNAoligonucleotides were synthesized by Invitrogen Inc., USA. Theoligonucleotides were deprotected and the complementary strands wereannealed.

SCA2-fibroblasts or -LB cells demonstrated increased abundance ofStaufen1 (FIGS. 3A-3G). The recapitulation of increased Staufen1 levelsby the expression of mutant ATXN2 in the BAC-SCA2 mouse model or in acell culture model, and dysregulation of RNA processing by theoverexpression of Staufen1 in cell culture (FIGS. 4A-4C and 5A-5F)predict that silencing of staufen1 might result in restoration of normallevels of Staufen1-target RNAs.

To test this, the consequences of PCP2 mRNA levels in SCA2-LB(ATXN2-Q22/52) cells in which staufen1 levels were reduced by siRNAdirected against Staufen1 were studied. qPCR analyses revealed thatdepletion of Staufen1 (˜83%) in SCA2-LB cells restored PCP2 mRNA levelto a level similar to that seen in normal LB cells treated with controlsiRNA (FIGS. 7A-7C). However, silencing of staufen1 (˜81%) in normal LBcells did not show significant alteration of PCP2 mRNA levels whencompared with normal LB cells treated with control siRNA (FIGS. 7A-7C).Of note, silencing of Staufen1 does not show any effect on ATXN2transcript levels in normal or SCA2-LB cells (FIG. 7D). Thus, silencingof Staufen1 rescued the effect of the mutant ATXN2 on aberrant RNAmetabolism observed in SCA2.

Next, mutant ATXN2 was depleted using siATXN2 RNA in a dose dependentfashion in SCA2-fibroblasts (ATXN2-Q22/42) or -LB (ATXN2-Q22/52) cells.Treatment with ATXN2 siRNA resulted in a dose-dependent reduction ofmutant ATXN2 in both cell types (FIGS. 7E, 7F, 7H, and 7I). Upondepletion of ATXN2, significant reduction of Staufen1 abundance and asignificant increase of PCP2 mRNA levels were observed in SCA2-LB cellsand -fibroblasts when compared with control siRNA (FIGS. 7E-7J). Thus,graded loss of ATXN2 lowered increased staufen1 abundance and resultedin restoration PCP2 mRNA levels in SCA2 cells.

Example 8—ASO Targeting Staufen1 Lowers Staufen1 Levels

The ATXN2 CAG22 repeat was edited to CAG58 repeats in the ATXN2 locus inHEK-293 cells using the CRISPR/Cas9/HDR (homology-directed-repair)technique. The human ATXN2 locus was engineered to replace CAG22 withCAG58 repeats in one ATXN2 allele. Modified CAG58 repeats flanking withlocus-specific 0.6 Kb left and right homologous arms (HAs) were clonedinto donor vector. The single guide RNA (sgRNA) that targeted ATXN2 wascloned into the hSpCas9-2A-Puro (PX459) vector (Addgene #62988). HEK-293cells were transfected with 1.0 μg of linearized donor vector and 1.0 μgof sgRNA and the pgRNA-Cas9 vector using the Lipofectamine 2000transfection reagent (ThermoFisher scientific). The cells were culturedwith 1.0 μg/ml puromycin for 7-10 days. The puromycin-resistant cellswere plated and cultured in a 96-well plate at 1 cell/well until wellswere ˜80% confluent with cells. The cells were then expanded andmaintained for PCR screening to identify knock-in positive cells.

HEK-293 cells were cultured in standard DMEM with 10% FBS and1×penicillin/streptomycin in 5% CO2. Transfection of STAU1 siRNA orSTAU1 ASO was accomplished using Lipofectamine transfection reagentusing standard conditions. Cells were harvested 3-5 days posttransfection.

Protein extracts were prepared by suspending cell pellets in SDS-PAGEsample buffer (Laemmli sample buffer) followed by boiling for 5 min.Equal amount of the extracts were subjected to Western blot analysis todetermine the steady-state levels of proteins using the indicatedantibodies. Protein extracts were resolved by SDS-PAGE and transferredto Hybond P membranes (Amersham Bioscience Inc., USA). After blockingwith 5% skim milk in 0.1% Tween 20/PBS, the membranes were incubatedwith primary antibodies in 5% skim milk in 0.1% Tween 20/PBS for 2 hrsat room temperature or overnight at 4° C. After several washes with 0.1%Tween 20/PBS, the membranes were incubated with the correspondingsecondary antibodies conjugated with HRP in 5% skim milk in 0.1% Tween20/PBS for 2 hrs at room temperature. Following three additional washeswith 0.1% Tween 20/PBS, signals were detected by using the ImmobilonWestern Chemiluminescent HRP Substrate (Millipore Inc., USA; cat#WBKLSO100) according to the manufacturer's protocol. The antibodiesused included ATXN2 mAb [(1:3000), BD Biosciences Inc.; cat #611378],rabbit anti-Staufen antibody (Novus biologicals Inc., NBP1-33202),SQSTM1/p62 antibody (Cell signaling, #5114), LC3B Antibody (Novusbiologicals Inc., NB100-2220), mTOR antibody (Cell signaling, #2972),Phospho-mTOR (Ser2448) antibody (Cell signaling #2971), 3-Actin mAbconjugated with HRP [(1:10,000), Sigma Inc.; cat #A3858]. Secondaryantibodies were horse anti-mouse IgG-HRP antibody (Vector laboratories,PI-2000), and goat anti-rabbit IgG-HRP antibody (Vector laboratories,PI-1000).

As illustrated in FIG. 8A, Staufen1 depletion restored autophagicpathway proteins in ATXN2-Q22/58 knock-in cells. Cells were transfectedwith STAU1 RNAi and analyzed by western blotting. Autophagy proteinsevaluated included the following: total (MTOR) and activated (pMTOR)mechanistic target of rapamycin which is an inhibitor of autophagy,SQSTM1/p62, or sequestosome-1, which binds proteins targeted to theautophagosome. LC3, a commonly used marker of autophagy function. LC3-Iis cytoplasmic while a cleaved form with higher mobility. LC3-II,localizes to the autophagosome. The amount of LC3-II is an indicator ofautophagosome quantity which typically increases when autophagy isdefective.

Further, as illustrated in FIGS. 8B and 8C, Antisense oligonucleotides(ASOs) targeting Staufen1 lower its expression in HEK-293-ATXN2[Q22/Q22]cells. STAU1 ASO-A (5′-TCTCATGTTGTAGTTATAGG-3′) (8B) (SEQ ID NO: 1) orSTAU1 ASO-B (5′-CTGGAAAGATAGTCCAGTTG-3′) (8C) (SEQ ID NO: 2) used at theindicated dose reduced STAU1 levels, determined by western blotting.Expression Relative to Actin.

Example 9—Staufen1 Links RNA Stress Granules and Autophagy inNeurodegeneration

DNA constructs. Human cDNA sequences for STAU1 (NM_001037328) werederived from the NCBI DNA database and used to design primers toPCR-amplify the coding sequences from a cDNA library made from HEK-293cells. All cDNAs including ATXN2 with expanded CAG repeats weresubsequently cloned into the appropriate vectors; pcDNA3.1/Flag (AgilentTechnologies, USA) plasmids. For in vitro RNA binding assay, humanStaufen1 and GFP coding sequences were PCR amplified and cloned intopET-His plasmids. All constructs were verified by sequencing.

siRNAs. The siRNAs used in this study were: All Star Negative ControlsiRNA (Qiagen Inc., USA; Cat #1027280), human siATXN2 [Hs_ATXN2_2(Qiagen Inc., USA; Cat #SI00308196)], human siSTA UI:5′-CCUAUAACUACAACAUGAGdTdT-3′ (SEQ ID NO: 41), and simTOR:5′-GAGCCUUGUUGAUCCUUAA-3′ (SEQ ID NO: 42). Staufen1 and mTOR siRNAoligonucleotides were synthesized by Invitrogen Inc., USA. Theoligonucleotides were deprotected and the complementary strands wereannealed.

Cell culture and Transfections. Four normal skin fibroblasts (ATXN2 withCAG22) and five SCA2 patient-derived skin FBs (ATXN2 with CAG repeats;35, 35, 35, 42, and 45), were maintained in DMEM medium containing 10%fetal bovine serum. Two normal (ATXN2 with CAG22) and three SCA2patient-derived (ATXN2 with expanded CAG repeats; 40, 46 and 52)Epstein-Barr virus (EBV)-immortalized LBCs were maintained in RPMImedium containing 15% fetal bovine serum. All subjects gave writtenconsent and all procedures were approved by the Institutional ReviewBoard at the University of Utah. The following primary human fibroblastswere obtained from the Coriell Cell Repositories (Camden, N.J., USA):ALS patient (TDP-43^(G298S) mutation) (ND32947), Huntington disease (HD)patient (ND33392) and SCA3 (Machado-Joseph disease, MJD) patient(GM06153). TDP-43^(G298S), HD, SCA3, and HEK-293 cells were maintainedin DMEM medium containing 10% fetal bovine serum.

For over-expression of recombinant proteins, HEK-293 cells were seededon 100 mm or 6 well dishes and incubated overnight. The cells were thentransfected with plasmid DNAs and harvested 48 hrs post-transfection andprocessed as two aliquots for protein and RNA analyses. For siRNAexperiments, cells were transfected with siRNAs using lipofectamine2000, and lymphoblastoid B cells (1×10⁶) were electroporated with siRNAsusing the Neon transfection system (Invitrogen Inc., USA) according tothe manufacturer's protocol and seeded on 6 well plates. Priorstandardization experiments showed that maximum silencing was achieved4-5 days post-transfection/electroporation.

Generation of HEK-293-ATXN2-Q22158 Knock-in (KI) cells.CRISPR/Cas9-mediated gene editing was carried out according to publishedprotocols. The human ATXN2 locus was engineered to replace CAG22 withCAG58 repeats in ATXN2 exon-1 in a BAC clone (RP11-798L5) containingATXV2 (Empire Genomics., USA). The modified CAG58 repeat with flankinglocus-specific 0.6 Kb left and right homologous arms (HAs) were clonedinto a donor vector. The sgRNA oligonucleotides targeting the ATXN2locus were annealed and cloned into the hSpCas9-2A-Puro (PX459) vector(Addgene #62988). HEK-293 cells were transfected with linearized donorvector and single guide RNA pgRNA-Cas9 vector using lipofectamine 2000transfection reagent (ThermoFisher scientific). The cells were culturedwith 1.0 μg/ml puromycin for ˜7-10 days. The puromycin-resistant cellswere plated and cultured in a 96-well plate at 1 cell/well until mostwells were ˜80% confluent with cells. The cells were then expanded andmaintained for PCR screening to identify knock-in positive cells.

Mice. ATXN2^(Q127) (Pcp2-ATXN2[Q127]) mice in a B6; D2 background, andBAC-SCA2 mice (BAC-Q22 and BAC-Q72) in an FVB background were used. TheStau1^(tm1Apa(−/−)) (Stau1^(−/−)) mouse was a generous gift from ProfMichael A. Kiebler, Ludwig Maximilian University of Munich, Germany, andmaintained in a B6 background. The TDP-43 transgene [B6;SJL-Tg(Thy1-TARDBP)4Singh/J] ALS mouse was purchased from the Jacksonlaboratory (Stock no. 012836) and maintained in a B6 background.Genotyping of animals was according to published protocols. All micewere bred and maintained under standard conditions consistent withNational Institutes of Health guidelines and conformed to an approvedUniversity of Utah IACUC protocol.

Antibodies. The antibodies used for western blotting andimmunohistochemistry and their dilutions were as follows: mouseanti-Ataxin-2 antibody (Clone 22/Ataxin-2) (BD Biosciences Inc.,611378), rabbit anti-neomycin phosphotransferase II (NPTII) antibody(EMD Millipore, AC113), rabbit anti-Staufen antibody (Novus biologicalsInc., NBP1-33202), rabbit anti-DDX6 antibody (Novus biologicals Inc.,NB200-191), RGS8 antibody (1:5000) (Novus Biologicals, NBP2-20153), LC3BAntibody (Novus biologicals Inc., NB100-2220), monoclonal anti-FLAG M2antibody (Sigma Inc., F3165), Monoclonal Anti-Calbindin-D-28K antibody[(1: 5000), Sigma Inc., C9848], monoclonal anti-β-Actin-peroxidaseantibody (clone AC-15) (Sigma Inc., A3854), goat anti-TIA-1 antibody(C-20) (Santa Cruz Inc., sc-1751), PCP-2 antibody (F-3) [(1: 3000),Santa Cruz Inc., sc-137064], Homer-3 Antibody (E-6) [(1: 2000), SantaCruz Inc., sc-376155], Anti-PCP4 antibody [(1: 5000) Abcam Inc.,ab197377], Anti-FAM107B antibody (1: 5,000) (Abcam Inc., ab175148),rabbit anti-PABP antibody (Abcam Inc., ab153930), p21 Waf1/Cip1 (12D1)rabbit mAb (Cell signaling, 2947), mTOR antibody (Cell signaling,#2972), Phospho-mTOR (Ser2448) antibody (Cell signaling #2971),SQSTM1/p62 antibody (Cell signaling, #5114), anti-c-Myc epitope tagmonoclonal antibody, HRP conjugate (Thermo Fisher Scientific, R951-21),6×-His epitope tag monoclonal antibody, HRP conjugate (HIS.H8)(Invitrogen Inc., MA1-21315-HRP) and sheep-anti-Digoxigenin-POD, Fabfragments (Roche Life Science, 11207733910). The secondary antibodieswere: horse anti-mouse IgG-HRP antibody (Vector laboratories, PI-2000),goat anti-rabbit IgG-HRP antibody (Vector laboratories, PI-1000) andhorse anti-goat IgG-HRP antibody (Vector laboratories, PI-9500).Fluorescent secondary antibodies were: goat anti-mouse IgG (H+L)antibody, DyLight-488 (Invitrogen Inc., 35502), goat anti-mouse IgG(H+L) antibody, DyLight-550 (Invitrogen Inc., 84540), goat anti-rabbitIgG, DyLight-549 (H&L) antibody (Thermo Scientific, 35557), goatanti-rabbit IgG (H+L) antibody, DyLight-488 (Invitrogen Inc., 35552),and donkey anti-goat IgG (H+L) antibody, DyLight-550 (Invitrogen Inc.,SA5-10087).

Immunofluorescence. Immunofluorescence studies were performed todetermine the co-localization of ATXN2 and Staufen1. Briefly, SCA2fibroblasts and HEK-293-ATXN2-Q22/58 KI cells were plated on coverslides overnight and fixed with 4% paraformaldehyde/PBS. To determinelocalization of ATXN2 with Staufen1 in stress granules, HEK-293 cellswere cultured on cover slides for overnight and heat-shocked at 43.5° C.for 1 hr. Cells were fixed with 4% paraformaldehyde/PBS, permeabilizedwith 0.1% Triton X-100, and processed for immunostaining usingcorresponding primary and fluorescent secondary antibodies. The nucleiwere stained with DAPI followed by mounting with Fluoromount-G (SouthernBiotech Inc.; 0100-01). The cells were imaged using confocal microscope(Nikon Eclipse Ti microscopy) and analyzed using the Nikon EZ-C1software. The co-localization plugin in ImageJ was used to defineco-localized areas via intensity-based thresholding. The AnalyzeParticles tool was then used to count the number of co-localized areasgreater than four pixels (baseline). Isolated cerebella were fixed in 4%paraformaldehyde and embedded in paraffin. Sections were deparaffinizedusing standard conditions, blocked with 5% donkey serum 0.3% TritonX-100 in PBS and processed for immunostaining. Primary antibodies:custom-designed ATXN2 rabbit polyclonal antibody [SCA2-280 (1:250)],STAU1 antibody (C-4) [(1:250), Santa Cruz Inc., sc-390820], andcorresponding fluorescent secondary antibodies were used. Images wereacquired using confocal microscope (Nikon Eclipse Ti microscopy) andanalyzed by the Nikon EZ-C1 software.

Preparation of protein lysates and western blot analyses. Cellularextracts were prepared by a single-step lysis method. The harvestedcells were suspended in SDS-PAGE sample buffer [Laemmli sample buffer(Bio-Rad Inc.; cat #161-0737)] and then boiled for 5 min. Equal amountsof the extracts were used for western blot analyses. Cerebellar proteinextracts were prepared by homogenization of mouse cerebella inextraction buffer [25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% NonidetP-40, 2 mM EDTA, 2 mM MgCl₂, 0.5 M urea and protease inhibitors (SigmaInc.; cat #P-8340)] followed by centrifugation at 4° C. for 20 min at14,000 RPM. Only supernatants were used for western blotting. Proteinextracts were resolved by SDS-PAGE and transferred to Hybond P membranes(Amersham Bioscience Inc., USA). After blocking with 5% skim milk in0.1% Tween 20/PBS, the membranes were processed for immunostaining usingcorresponding primary and secondary antibodies. Signals were detected byusing the Immobilon Western Chemiluminescent HRP Substrate (MilliporeInc., USA; cat #WBKLSO100) according to the manufacturer's protocol. Theintensity of proteins was determined by using the ImageJ softwareanalyses system and the relative protein abundances were expressed asratios to 3-actin.

Immunoprecipitations. To determine protein-protein or protein-RNAinteractions, we carried out protein-RNA immunoprecipitation (IP)experiments using HEK-293 cells expressing Flag-ATXN2-Q22 andFlag-ATXN2-Q108 or Flag-Staufen1. The preparation of whole cell extractsand IP methods followed previously published methods. First, cells werelysed with a cytoplasmic extraction buffer [25 mM Tris-HCl pH 7.6, 10 mMNaCl, 0.5% NP40, 2 mMEDTA, 2 mM MgCl₂, protease inhibitors (Sigma Inc.;Cat #P-8340)] and cytoplasmic extracts were separated by centrifugationat 14,000 RPM for 20 min. Second, the resultant pellets were suspendedin nuclear lysis buffer or high salt lysis buffer (25 mM Tris-HCl, pH7.6, 500 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl₂, proteaseinhibitors), and the nuclear extracts were separated by centrifugationat 14,000 RPM for 20 min. The nuclear extracts were then combined withthe cytoplasmic extracts and denoted whole cell extracts. Specifically,while combining cytoplasmic and nuclear extracts, the NaCl concentrationwas adjusted to physiologic buffer conditions (˜150 mM) to preserve invivo interactions. For identifying non-RNA mediated interactions, wholecell extracts were treated with 1.0 mg/ml RNase A (Amersham BioscienceInc., USA) for 15 min at 37° C. before subjected to IP. Ninety percentof cell extracts were subjected to Flag monoclonal antibody (mAb) IP(Anti-FlagM2 Affinity Gel, Sigma Inc.; cat #A2220-1ML) toimmunoprecipitate ATXN2 or Staufen1 interacting protein-RNA complexes.The remaining 10% of whole cell extracts were saved as the input controlfor western blotting and RT-PCR analyses. The IPs were washed with abuffer containing 200 mM NaCl and the bound protein-protein orprotein-RNA complexes were eluted from the beads with Flag peptidecompetition (100 μg/ml). Eluted fractions were divided into two equalparts. One part was analyzed by SDS-PAGE followed by western blotting todetermine interactions between ATXN2 and Staufen1. RNA was isolated fromthe other fraction and subjected to RT-PCR analyses to identify RNAsthat bound to Staufen1.

In vitro RNA binding (Northwestern) assay. Northwestern blot assays wereperformed following a published protocol with some modifications.BL21<DE3> cells carrying His-Staufen1, His-GFP and empty pET vectors,were grown to mid-log phase. Whole cell lysates from harvested cellswere run on SDS-PAGE and electro-blotted onto a Hybond P membrane. Thetransferred proteins were re-natured as follows: the blot was firstincubated with binding buffer (0.1 M HEPES, pH 7.9, 0.1 M MgCl₂, 0.1 MKCl, 0.5 μM ZnSO₄) with 1 mM DTT and 6 M urea for 5 min at roomtemperature. The blots were then incubated, for 5 min each, through fiveserial twofold dilutions of urea with binding buffer/1 mM DTT withcontinuing incubation steps until the binding buffer was 1 mM DTTwithout urea. Blot pre-hybridizations were carried out with bindingbuffer (1 mM DTT, 5% BSA, 1 μg/ml yeast tRNA) for 1 hr at roomtemperature. The blots were then hybridized with DIG-labelled RNA probesin binding buffer (1 mM DTT, 0.25% BSA, 1 μg/ml yeast tRNA) overnight at4° C. After several washes with 0.1% Tween 20/PBS, the membranes wereincubated with anti-DIG-POD antibody in 5% skim milk in 0.1% Tween20/PBS for 2 hr at room temperature. Following three additional washeswith 0.1% Tween 20/PBS, signals were detected by using the ImmobilonWestern Chemiluminescent HRP Substrate according to the manufacturer'sprotocol.

RNA expression analyses by Quantitative RT-PCR. Mice were deeplyanesthetized with isoflurane. Mouse cerebella were removed andimmediately submerged in liquid nitrogen. Tissues were kept at −80° C.until the time of processing. Total RNA was extracted from mousecerebella and from harvested cells using the RNaeasy mini-kit accordingto the manufacturer's protocol (Qiagen Inc., USA). DNAse I treated RNAswere used to synthesize cDNAs using the ProtoScript cDNA synthesis kit(New England Biolabs Inc., USA). Quantitative RT-PCR was performed inQuantStudio 12K (Life Technologies, Inc.; USA) with the Power SYBR GreenPCRMaster Mix (Applied Biosystems Inc.; USA). PCR reaction mixturescontained SYBR Green PCRMaster mix, synthesized cDNA and 0.5 pmolprimers, and PCR amplifications were carried out for 45 cycles:denaturation at 95° C. for 10 sec, annealing at 60° C. for 10 sec andextension at 72° C. for 40 sec. The threshold cycle for each sample waschosen from the linear range and converted to a starting quantity byinterpolation from a standard curve run on the same plate for each setof primers. All gene expression levels were normalized to the ACTB orGAPDH or Actb mRNA levels. Primer pairs designed for qRT-PCR and RT-PCRare given as forward and reverse, respectively, and listed in Table 2below.

TABLE 2 RT-PCR and qPCR primer sequences SCA2-A 5′-GGGCCCCTCACCATGTCG-3′Human RT-PCR SEQ ID NO: 43 SCA2-B 5′-CGGGCATGCGGACATTGG-3′ Human RT-PCRSEQ ID NO: 44 ATXN2- 5′-CCGCCCGGCGTGCGAGCCGGTGTATGG-3′ Human RT-PCRSEQ ID NO: Exon1-F 45 ATXN2- 5′-GTAGACTGAGGCAGTCCTTTGTTACTG-3′ HumanRT-PCR SEQ ID NO: Exon2-R 46 GAPDH-F 5′-ACATCGCTCAGACACCATG-3′ Human RT-SEQ ID NO: PCR/qRT- 47 PCR GAPDH-R 5′-TGTAGTTGAGGTCAATGAAGGG-3′ HumanRT- SEQ ID NO: PCR/qRT- 48 PCR ACTB-F 5′-GAAAATCTGGCACCACACCT-3′ HumanqRT-PCR SEQ ID NO: 49 ACTB-R 5′-TAGCACAGCCTGGATAGCAA-3′ Human qRT-PCRSEQ ID NO: 50 STAU1-F 5′-TCCATGGTTTCAAAGTCCCG-3′ Human qRT-PCRSEQ ID NO: 51 STAU1-R 5′-ATTTTCATCCCCAGAGCCAG-3′ Human qRT-PCRSEQ ID NO: 52 PCP2-F 5′-AAGGACGGAGCACAGAAAC-3′ Human qRT-PCR SEQ ID NO:53 PCP2-R 5′-GAGTGAGACCCAGGATGC-3′ Human qRT-PCR SEQ ID NO: 54 ATXN2-F5′-AAGATATGGACTCCAGTTATGCAAA-3′ Human qRT-PCR SEQ ID NO: 55 ATXN2-R5′-CAAAGCCTCAAGTTCCTCAT-3′ Human qRT-PCR SEQ ID NO: 56 CACNA1G5′-CCGACCCACAGATCCCTCTA-3′ Human qRT-PCR SEQ ID NO: 57 CACNA1G5′-GCTGTCATTGGGCAGAGAGT-3′ Human qRT-PCR SEQ ID NO: 58 ITPR15′-GCACGTCTTCCTGAGAACCA-3′ Human qRT-PCR SEQ ID NO: 59 ITPR15′-CACTGAGGGCTGAAACTCCA-3′ Human qRT-PCR SEQ ID NO: 60 PCP2-F5′-GCCAGATCCAGCATCGTTGT-3′ Human RT-PCR SEQ ID NO: 61 PCP2-R5′-CTCTGGCTCTTGGTGGTCTG-3′ Human RT-PCR SEQ ID NO: 62 mTOR-175′-CGAACCTCAGGGCAAGATG-3′ Human RT-PCR SEQ ID NO: 63 mTOR-R5′-TTTCCTCATTCCGGCTCTTTAG-3′ Human RT-PCR SEQ ID NO: 64 Stau1-F5′-AGTACATGCTCCTTACAGAACG-3′ Mouse qRT-PCR SEQ ID NO: 65 Stau1-R5′-TGATGCCCAACCTTTACCTG-3′ Mouse qRT-PCR SEQ ID NO: 66 Actb-F5′-CGTCGACAACGGCTCCGGCATG-3′ Mouse qRT-PCR SEQ ID NO: 67 Actb-R5′-GGGCCTCGTCACCCACATAGGAG-3′ Mouse qRT-PCR SEQ ID NO: 68

Rotarod Testing. Following genotyping mice were randomly assigned tocages according to sex, ensuring that each possible genotype wasrepresented in each cage. During the five days of testing, mice weretaken to a separate testing room and allowed to habituate for one hour.Testing began at the same time every day and was completed by the sametechnician. The technician was blinded to the genotypes of the mice ineach cage. On day 1 mice were handled for 2 min per mouse. On day 2 micewere placed on an accelerating rotarod apparatus (Rotamex-5, ColumbusInstruments, Columbus, Ohio, USA) initially rotating at 4 RPM for 2 min.The rate of rotation was increased by 1 RPM per every 15 s to 10 RPM for60 s. On days 3-5, mice were tested on the accelerating rotarod from 0RPM with acceleration increasing by 1 RPM every 9 s until the maximumspeed of 51 RPM was reached (maximum time of 459 s). The time that amouse fell (latency to fall) from the rotating bar was recorded.Statistical comparisons of rotarod data were determined using the methodof generalized estimating equations (GEE) with the independentcorrelation option using Stata 12 (procedures xtset followed by xtgee).

Statistical analysis. For western blot analyses, the experiments wereperformed three times, and wherever appropriate, gels were scanned andband intensities quantified by ImageJ analyses after inversion of theimages. The P values were calculated by pairwise Student's t-test todetermine whether difference between groups were significant. The levelof significance was set at P<0.05. In the figures, a single asteriskindicates P<0.05, a double asterisk P<0.01, a triple asterisk P<0.001,and ns represents P>0.05 (Student's t-test).

Results. Disorders of RNA metabolism have recently come into focuseither as directly causative via mutation of the respective proteins oras contributing through formation of RNA stress granules andsequestration of proteins into these granules. Thus, mutations in theRNA binding protein, Tar DNA binding protein-43 (TDP-43) cause ALS andALS/Frontotemporal lobar degeneration (FTLD), but other genetic ornon-genetic forms of ALS also show TDP-43 accumulation in spinal motorand cortical neurons.

To further define components of the dysregulated stress response in SCA2and TDP43-mediated motor neuron death the ATXN2 protein complex waspurified. As described above, a prominent protein in the ATXN2 complexin HEK-293 cells is STAU1, showing RNA-dependent interaction withwild-type or mutant ATXN2 by co-immunoprecipitation (FIG. 1F). STAU1 isa well-characterized double-stranded RNA-binding protein and componentof RNA stress granules (SGs). As also described above, heat-stressedHEK-293 cells produced SGs positive for both ATXN2 and STAU1 (FIG. 1A).In SCA2 patient fibroblasts (FBs), constitutively-present ATXN2/STAU1SGs were observed even in the absence of stress as well as in Purkinjecells of SCA2 (ATXN2^(Q127)) mice (FIGS. 1B-1C and FIGS. 9A-9B). Uponverification of the levels of proteins in SCA2-patient FBs andlymphoblastoid cells (LBCs), it was noted that STAU1 levels weresignificantly increased in the presence of endogenous mutant ATXN2(FIGS. 10A-10B). This observation was also confirmed by analysis ofcerebellar extracts from two animal models of SCA2 (ATXN2^(Q127) andBAC-Q72) (FIGS. 10C-10D).

The sensitivity of STAU1 to cellular stress prompted the examination ofwhether this pathway was active in other neurodegenerative conditions.Protein extracts were prepared from cerebella and spinal cords fromanimals overexpressing wild-type TDP-43, a mouse model of ALS. For theseexperiments, hemizygous transgenic animals were used that develop aphenotype late, at 15 months, facilitating testing Stau1 levels severalmonths prior to onset of symptoms. There were significant increases inStau1 and wild-type Atxn2 in both cerebellum and spinal cord (FIGS.10E-10F), aligning with improved TDP-43 mouse survival with therapeuticreduction of wild-type Atxn2. STAU1 levels were also increased inALS-patient FBs harboring the TDP-43^(G298S) mutation (FIG. 10G). STAU1levels were then determined in cell lines from patients with Huntingtondisease (HD) or SCA3. At physiologic conditions, STAU1 levels were notdifferent in HD and SCA3 fibroblasts as compared with normalfibroblasts. When sodium arsenite was added to cultures, however, cleardifferences emerged. While only a modest STAU1 increase in response toarsenite was detected and only visible after 6 hr treatment, patientcells showed early and exaggerated responses to arsenite indicatinghypersensitivity of the STAU1 pathway in these cells (FIG. 10H).

As stress granules are degraded by autophagy, it was examined whetherincreased STAU1 levels were due to slowed turnover. STAU1 mRNA levelswere not increased in patient cell lines and rodent cerebella by qRT-PCR(FIGS. 11A-11D), but STAU1 degradation was slowed, determined by acycloheximide (CHX) pulse-chase experiment (FIG. 12 and FIG. 4C).Indeed, analysis of autophagic flux in SCA2 patient cells indicated thatautophagy was impaired in the presence of mutant ATXN2 (FIG. 13A). Inorder to investigate STAU1 directly in autophagy, CRISPR/Cas9 genomeediting was used to introduce an expanded CAG⁵⁸ (Q58) repeat into oneATXN2 allele in human HEK-293 cells. These cells were designatedATXN2-Q22/58 knock-in (KI) cells. ATXN2-Q22/58 KI cells had elevatedSTAU1 abundance, and ATXN2/STAU1 SGs (FIG. 13C and FIGS. 14A-14D). Inprior studies, key transcriptomic changes were identified in SCA2 mousemodels including reduced abundances in cerebellar PCP2, CACNA1G, andITPR1 mRNAs. These changes were replicated in HEK-293 ATXN2-Q22/58 KIcells in the presence of one mutant ATXN2 allele (FIGS. 14A-14D).

Compared with wild-type HEK-293 cells, the presence of endogenous mutantATXN2 significantly increased abundance of active mechanistic target ofrapamycin (mTOR), sequestosome 1 (SQSTM1/p62) and processed LC3-II (thelipidated isoform of LC3-I) (FIG. 13C). It was verified that the sameautophagy changes including STAU1 abundance occurred in HEK-293 cellsfollowing overexpression of ATXN2 harboring Q58 and Q108 repeats but notQ22 (FIG. 15A). Consistent with inhibited autophagy, we showed thatHEK-293 cells treated with bafilomycin A1 (Baf), an inhibitor ofautophagosome-lysosome fusion, expressed p62 and processed LC3-IIsimilar to cells expressing ATXN2-Q58 (FIG. 15B). However, the slightelevation of LC3-II with Baf treatment suggested that partial autophagycompetence persisted in ATXN2-Q22/58 KI cells.

To test whether changes in autophagy were largely due to increased STAU1levels, STAU1 was exogenously expressed in HEK-293 cells. This resultedin increased expression of activated P-mTOR, mTOR, p62 and LC3-II, aswell as reduction of the STAU1 target, PCP2 protein (FIG. 15C).Rapamycin- or rapalog-induced mTOR signaling inhibition has been shownto induce autophagy, reduce polyglutamine toxicity and motor deficits inHD models, and ameliorate motor phenotypes in mice modeling autismspectrum disorders (ASDs). Indeed, it was demonstrated that STAU1 levelsand the abnormal levels of activated P-mTOR, mTOR, p62, and LC3-II wererestored by rapamycin treatment, as well as by lowering either STAU1,ATXN2, or mTOR by RNAi in ATXN2-Q22/58 KI cells (FIGS. 16A-16B and FIGS.17A-17B). These experiments also showed that STAU1 acted downstream ofATXN2 as ATXN2-RNAi reduced STAU1, but STAU1-RNAi did not change ATXN2levels. Furthermore, STAU1 and mTOR functionally interact in areciprocal fashion as RNAi to either one can normalize the level of theother protein.

One of the functions of STAU1 is targeting subsets of mRNAs fordegradation via a mechanism similar to nonsense-mediated decay, referredto as STAU1-mediated RNA decay (SMD). In genome-wide transcriptomeanalysis in SCA2 animal models, it was noticed that a large number ofmRNAs were severely decreased in abundance. Some of these RNAs had alsobeen identified as STAU1 interactors by hiCLIP. SMD for a given mRNApredicts that STAU1 binds to the 3′UTR of the respective mRNA.

To test SMD for PCP2 mRNA, STAU1 was shown to bind PCP2 directly, andthat binding was abolished in an mRNA lacking the 3′UTR (FIG. 13B andFIGS. 6B-6C). This is further supported by restoration of PCP2expression in SCA2-patient LBCs after reduction of STAU1 via RNAi, andby the ability for STAU1 to lower PCP2 expression by way of aninteraction with the PCP2 3′UTR (FIGS. 18A-18C and FIGS. 19A-19B). SMDmay explain increased RNA abundance for a subset of mRNAs identified inSCA2 patient cells and animal models by STAU1 binding to the 3′UTRs ofthe respective mRNAs. STAU1 also regulates the translational efficiencyvia 5′UTR and polysome association³. Consistent with this, interactionbetween Staufen1 and a 5′UTR-mTOR RNA by STAU1 immunoprecipitation wasshown (FIG. 13B), supporting its role in mTOR translation.

To test targeting Stau1 in vivo, we used a well characterized model ofcerebellar neurodegeneration expressing mutant ATXN2^(Q127) in PCs.ATXN2^(Q127) mice develop progressive behavioral and proteomic deficitswith an onset at 8 weeks. We crossed these mice with a previouslygenerated mouse line deficient for one Stau1 allele. Reducing Stau1dosage by 50% led to a significant improvement of motor dysfunction ofATXN2^(Q127) mice as measured by performance on the accelerating rotarod(FIG. 16C). Although Stau1 reduction was present in the germline, thusprior to symptom onset, the effect on motor performance was onlydiscernible after 8 weeks. This may suggest that Staufen1 reduction mayexert its major effect in neurons experiencing chronic stress.Significant and progressive decreases in key Purkinje cell mRNAs andeven greater decreases in the respective encoded proteins (Calb1, Pcp2,Pcp4, Rgs8, Homer3 and Fam107b) have been described in ATXN2^(Q127)mice. Reduction of Stau1 expression by 50% in vivo significantlyincreased levels of these six proteins towards normalization. Uponcomplete genetic ablation of Stau1 in ATXN2^(Q127) mice, the levels ofthese proteins returned to normal (FIGS. 16D-16F and FIG. 20).

It is noteworthy that—similar to our in vitro studies—STAU1 ablation invivo had only minimal effects in wild-type animals. The levels ofprotein significantly impacted by absence of STAU1 in mutant transgenicanimals remained unchanged in wild-type animals. The same was true formarkers of autophagy (FIGS. 16D-16F and FIG. 20). This suggests thatSTAU1 serves as a sensor of cellular stress and once engaged, showsrapid amplification of the signal via reciprocal effects on mTOR andautophagy. In the absence of stress, changes of STAU1 levels appear tohave relatively minor effects at least within the parameters measured inthis study.

Example 10—STAU1 in ALS cells and Alzheimer's disease and its relateddementias (AD/ADRD)

ALS patient fibroblasts with TDP-43(G298S) mutations had increased STAU1(FIG. 21A). HEK293 cells transfected with TDP-43 or mutant forms ofTDP-43 [TDP-43(G298S), TDP-43(G348C) and TDP-43(A382T) or truncatedTDP-25] had increased STAU1 and LC3-II levels (FIGS. 21B-21D).

STAU1 abundance was also elevated in cells with GGGGCC repeat expansionsin C9ORF72-ALS (FIG. 22A). In another experiment, non-mutant C9ORF72were overexpressed and elevated STAU1 and LC3-II were observed (FIG.22B). Also, STAU1 was observed to be overabundant in fibroblasts from aPD patient with a MAPT (N279K) mutation (FIG. 22A) and in HEK293 cellsoverexpressing GFP-TAU with a P301L mutation causing frontotemporaldementias (FTD), and an amyloid precursor protein (APP) with theSwedish/Indiana mutations causative for familial AD (FIG. 22C).

Example 11—Relationship Between Staufen and Autophagy

Methods DNA constructs. The plasmid constructs used in this study were3XFlag-tagged STAU1 or wild-type TDP-43, His-tagged STAU1 or GFP andpRK5-EGFP-TAU (Addgene, Plasmid #46904). Mutant TDP-43 cDNAs(TDP-43^(G298S) and TDP-43^(A382T)) were PCR-amplified from cDNAlibraries of ALS patient FBs with TDP-43 mutations (G298S #ND32947 andA382T #ND41003) (Coriell Cell Repositories, Camden, N.J., USA).TDP-43^(G348C), C-terminal fragment of TDP-43 (TDP-43^(CTF)) andRNA-binding domain 3 deletion of STAU1 (STAU1[RBDΔ3]) constructs weregenerated using TDP-43 and STAU1 cDNAs as templates, respectively. ThePCR products were cloned into pCMV-3XFlag plasmid (Agilent Technologies,USA). All constructs were verified by sequencing. 3XFlag is referred toas Flag in the text and figures.

siRNAs and Reagents. The siRNAs used in this study were: All StarNegative Control siRNA (Qiagen, Cat #1027280) and human siSTAU1:5′-CCUAUAACUACAACAUGAGdTdT-3′. Staufen1 siRNA oligonucleotides weresynthesized by Invitrogen, USA. The oligonucleotides were deprotectedand the complementary strands were annealed. Thapsigargin (Tocris USA,Cat #1138), Tunicamycin (Tocris USA, Cat #3516), Ionomycin (Tocris USA,Cat #1704) and Sodium arsenite solution (Sigma-Aldrich, Cat #1062771000)were used in this study.

Cell culture and Transfections. Five SCA2 patient-derived skin FBs(ATXN2 with CAG repeats; 35, 35, 35, 42, and 45) were maintained in DMEMmedium containing 10% fetal bovine serum. All subjects gave writtenconsent and all procedures were approved by the Institutional ReviewBoard (IRB) at the University of Utah. The following primary humanfibroblasts were obtained from the Coriell Cell Repositories (Camden,N.J., USA): Normal FBs (#ND29510, #ND34769 and #ND38530), two ALSpatients with TDP-43 mutations (G298S #ND32947 and A382T #ND41003),three ALS patients with C9orf72 mutations (#ND40069, #ND42504 and#ND42506), three Huntington disease (HD) patients (Q44 #ND31038, Q57#ND33392 and Q66 #ND40536) and four Alzheimer's disease (AD) patients[(PSEN1 mutations: M146I #ND34730, E184D #ND34732 and Intron4 Var#ND41001) and MAPT mutation: N279K #ND40074]. All FBs includingHEK-293-ATXN2-Q22/58 knock-in (KI) cells3 used in this study weremaintained in DMEM medium containing 10% fetal bovine serum.

For over-expression of recombinant proteins, HEK-293 cells were seededon 6 well dishes and incubated overnight. The cells were thentransfected with plasmid DNAs and harvested 48 hrs post-transfection andprocessed as two aliquots for protein and RNA analyses. For siRNAexperiments, cells were transfected with siRNAs using lipofectamine 2000transfection reagent (ThermoFisher Scientific) according to themanufacturer's protocol. Prior standardization experiments showed thatmaximum silencing was achieved 4-5 days post-transfection.

Cell line authentication. Cell lines used in the study (as HEK-293 cellsor human fibroblasts, etc) were authenticated by short tandem repeat(STR) analysis, an approach that evaluates 24 loci including Amelogeninfor sex identification, using the GenePrint 24 System (Promega, USA).PCR sequencing was also used to verify the presence of mutations inpatient-derived cells. This is in part in response to NIH guidelines onscientific rigor in conducting biomedical research (NOT-OD-15-103).

Mice. ATXN2^(Q127) (Pcp2-ATXN2[Q127]) mice were maintained in a B6D2F1/Jbackground. The Stau1^(tm1Apa(−/−)) (Stau1^(−/−)) mouse was a generousgift from Prof. Michael A. Kiebler, Ludwig Maximilian University ofMunich, Germany, and maintained in a C57BL/6J background.

ATXN2^(Q127) mice were crossed with Stau1^(−/−) mice to generateATXN2^(Q127); Stau1^(+/−) and ATXN2; Stau1^(+/−) mice. These mice werethen interbred to generate ATXN2^(Q127); Stau1^(−/−) and ATXN2^(WT);Stau1^(−/−) mice. These mice were in a mixed background of B6D2F1/JC57BL/6J. B6; SJL-Tg(Thy1-TARDBP)4Singh/J (Stock 012836) purchased fromJackson Laboratories. Jackson Laboratories B6SJLF1/J mice (Stock No.100012) were backcrossed to C57BL/6J to N2 before crossing them toStau1^(−/−) mouse. Genotyping of animals was accomplished according topublished protocols. All mice were bred and maintained under standardconditions consistent with National Institutes of Health guidelines andconformed to an approved University of Utah IACUC protocol. Wild-typeand BAC-C9orf72 mouse brain extracts were provided by Laura P. W. Ranum,Department of Neurology, University of Florida, Gainesville, Fla. 32610,USA.

Primary culture of cortical neurons. Primary cortical neuron cultureswere prepared from neonatal wild-type or Stau1^(−/−) mice(Stau1^(tm1Apa(−/−))). Brain cortices from 6-7 animals were isolated andincubated with 50 units of papain (Worthington Biochemical, USA) inEarle's balanced salt solution (EBSS) with 1.0 mM L-cysteine and 0.5 mMEDTA for 15 minutes at 37° C., followed by washing in EBSS andmechanical trituration. The remaining tissues were removed by filtrationthrough a 50 μm strainer (Falcon). Neurons were seeded at 50×10³ per cm²on plates coated with poly-L-omithine and laminin in Neurobasal Plusmedium containing 2% B27 Plus supplement (Life technologies, USA). Onday 2, 10 μM cytosine arabinoside was added for 24 hr to preventproliferation of glial cells and 75% of culture medium volume wasreplenished every 2-3 days from there on. Experiments were conducted onday 9-10 by replacing all culture medium with fresh media containingthapsigargin or vehicle (DMSO), and 18 hr later protein lysates wereprepared following standard procedures.

Antibodies. The antibodies used for western blotting and their dilutionswere as follows: mouse anti-Ataxin-2 antibody (Clone 22/Ataxin-2)[(1:4000), BD Biosciences, Cat #611378], rabbit anti-Staufen antibody[(1:5000), Novus biologicals, NBP1-33202], LC3B Antibody [(1:8000),Novus biologicals, NB100-2220], TDP-43 antibody [(1:8000), Proteintech,Cat #10782-2-AP], monoclonal anti-FLAG M2 antibody [(1:10,000),Sigma-Aldrich, F3165], monoclonal anti-β-Actin-peroxidase antibody(clone AC-15) [(1:30,000), Sigma-Aldrich, A3854], SQSTM1/p62 antibody[(1:4000), Cell Signaling, Cat #5114], mTOR antibody [(1:4000), CellSignaling, Cat #2972], Phospho-mTOR (Ser2448) antibody [(1:3000), CellSignaling, Cat #2971], p70 S6 Kinase antibody [(1:4000), Cell Signaling,Cat #9202], Phospho-p70 S6 Kinase (Thr389) antibody [(1:3000), CellSignaling, Cat #9205], 4E-BP1 antibody [(1:4000), Cell Signaling, Cat#9452], Phospho-4E-BP1 (Thr37/46) (236B4) rabbit mAb [(1:3000), CellSignaling, Cat #2855], 6×-His Tag monoclonal antibody (HIS.H8), HRP[(1:10,000) (ThermoFisher Scientific, MA1-21315-HRP)] andsheep-anti-Digoxigenin-POD, Fab fragments [(1:10,000), Roche LifeScience, Cat #11207733910]. The secondary antibodies were:Peroxidase-conjugated horse anti-mouse IgG (H+L) antibody [(1:5000),Vector laboratories, PI-2000] and Peroxidase-conjugated AffiniPure goatanti-rabbit IgG (H+L) antibody [(1:5000), Jackson ImmunoResearchLaboratories, Cat #111-035-144].

Preparation of protein lysates, western blotting and in vitro RNAbinding assays. Cellular extracts were prepared by a single-step lysismethod. The harvested cells were suspended in SDS-PAGE sample buffer[Laemmli sample buffer (Bio-Rad, Cat #161-0737)] and then boiled for 5min. Equal amounts of the extracts were used for western blot analyses.

Cerebellar or spinal cord protein extracts were prepared byhomogenization of mouse tissues in extraction buffer [25 mM Tris-HCl pH7.6, 300 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl₂, 0.5 M ureaand protease inhibitors (Sigma-Aldrich, P-8340)] followed bycentrifugation at 4° C. for 20 min at 14,000 RPM. Only supernatants wereused for western blotting. Protein extracts were resolved by SDS-PAGEand transferred to Hybond P membranes (Amersham Bioscience, USA), andthen processed for western blotting according to our published protocol.Signals were detected by using the Immobilon Western ChemiluminescentHRP Substrate (EMD Millipore, WBKLSO100) according to the manufacturer'sprotocol. Blot images were scanned and band intensities were quantifiedby ImageJ software analyses after inversion of the images, and therelative protein abundances were expressed as ratios to 3-Actin. Invitro RNA binding (Northwestern) assays were performed to determineSTAU1 and mTOR-5′UTR interaction using a previously described protocol.

Immunoprecipitations. To determine protein-RNA interactions, protein-RNAimmunoprecipitation (IP) experiments were carried out using HEK-293cells expressing Flag-STAU1. The preparation of whole cell extracts andIP methods followed previously published methods. First, cells werelysed with a cytoplasmic extraction buffer [25 mM Tris-HCl pH 7.6, 10 mMNaCl, 0.5% NP40, 2 mMEDTA, 2 mM MgCl₂, protease inhibitors] andcytoplasmic extracts were separated by centrifugation at 14,000 RPM for20 min. Second, the resultant pellets were suspended in nuclear lysisbuffer or high salt lysis buffer (25 mM Tris-HCl, pH 7.6, 500 mM NaCl,0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl₂, protease inhibitors), and thenuclear extracts were separated by centrifugation at 14,000 RPM for 20min. The nuclear extracts were then combined with the cytoplasmicextracts and denoted whole cell extracts. Specifically, while combiningcytoplasmic and nuclear extracts, the NaCl concentration was adjusted tophysiologic buffer conditions (˜150 mM) to preserve in vivointeractions. Ninety percent of cell extracts were subjected to Flagmonoclonal antibody (mAb) IP (Anti-FlagM2 Affinity Gel, Sigma-Aldrich,A2220-1ML) to immunoprecipitate STAU1 interacting protein-RNA complexes.The remaining 10% of whole cell extracts were saved as the input controlfor western blotting and RT-PCR analyses. The IPs were washed with abuffer containing 200 mM NaCl and the bound protein-RNA complexes wereeluted from the beads with Flag peptide competition (100 μg/ml) (FlagPeptide, Sigma-Aldrich, F3290-4MG). Eluted fractions were divided intotwo equal parts. One part was analyzed by SDS-PAGE followed by westernblotting to determine STAU1 expression. RNA was isolated from the otherfraction and subjected to RT-PCR analyses to identify RNAs that bound toSTAU1.

RNA expression analyses by quantitative RT-PCR. Mice were deeplyanesthetized with isoflurane then decapitated. Mouse cerebella or spinalcord were removed and immediately submerged in liquid nitrogen. Tissueswere kept at −80° C. until the time of processing. Total RNA wasextracted from mouse tissues and harvested cells using the RNeasyMini-Kit according to the manufacturer's protocol (Qiagen, USA). DNAse Itreated RNAs were used to synthesize cDNAs using the ProtoScript cDNAsynthesis kit (New England Biolabs, USA). Quantitative RT-PCR wasperformed in QuantStudio 12K (Life Technologies, USA) with the PowerSYBR Green PCRMaster Mix (Applied Biosystems, USA) using University ofUtah genomics core lab. PCR reaction mixtures contained SYBR GreenPCRMaster mix, synthesized cDNA and 0.5 pmol primers, and PCRamplifications were carried out for 45 cycles: denaturation at 95° C.for 10 sec, annealing at 60° C. for 10 sec and extension at 72° C. for40 sec. The threshold cycle for each sample was chosen from the linearrange and converted to a starting quantity by interpolation from astandard curve run on the same plate for each set of primers. CommercialTaqMan assay (Cat #4331182: Hs00244999_m1 for STAU1 probe andHs01060665_g1 for ACTB probe, ThermoFisher Scientific) was used todetect STAU1. All gene expression levels were normalized to ACTB orGAPDH or Actb mRNA levels. Primer pairs designed for RT-PCR and qRT-PCRare given as forward and reverse, respectively, and listed in Table 3below.

TABLE 3 RT-PCR and qRT-PCR primer sequences GAPDH-F5′-ACATCGCTCAGACACCATG-3′ Human RT-PCR SEQ ID NO: 47 GAPDH-R5′-TGTAGTTGAGGTCAATGAAGGG-3′ Human RT-PCR SEQ ID NO: 48 mTOR-F5′-CGAACCTCAGGGCAAGATG-3′ Human RT-PCR SEQ ID NO: 63 mTOR-R5′-TTTCCTCATTCCGGCTCTTTAG-3′ Human RT-PCR SEQ ID NO: 64 ACTB-F5′-GAAAATCTGGCACCACACCT-3′ Human qRT-PCR SEQ ID NO: 49 ACTB-R5′-TAGCACAGCCTGGATAGCAA-3′ Human qRT-PCR SEQ ID NO: 50 mTOR-F5′-CAGAAGGTGGAGGTGTTTGAG-3′ Human qRT-PCR SEQ ID NO: 69 mTOR-R5′-TGACATGACCGCTAAAGAACG-3′ Human qRT-PCR SEQ ID NO: 70 Stau1-F5′-AGTACATGCTCCTTACAGAACG-3′ Mouse qRT-PCR SEQ ID NO: 65 Stau1-R5′-TGATGCCCAACCTTTACCTG-3′ Mouse qRT-PCR SEQ ID NO: 66 mTor-F5′-ATTCAATCCATAGCCCCGTC-3′ Mouse qRT-PCR SEQ ID NO: 71 mTor-R5′-TGCATCACTCGTTCATCCTG-3′ Mouse qRT-PCR SEQ ID NO: 72 Actb-F5′-CGTCGACAACGGCTCCGGCATG-3′ Mouse qRT-PCR SEQ ID NO: 67 Actb-R5′-GGGCCTCGTCACCCACATAGGAG-3′ Mouse qRT-PCR SEQ ID NO: 68Statistical analysis. Student's t-tests were used to determine whetherdifferences between groups were significant. The level of significancewas set at P≤0.05. *P≤0.05, **P≤0.01, ***P≤0.001 and ns=P>0.05. Means±SDare presented throughout, unless otherwise specified.

Results

STAU1 overabundance is common in cell lines derived from patients withneurodegenerative diseases. Further research was performed to determinewhether STAU1 overabundance represented a more generalized response ofcells stressed by the presence of mutant disease proteins or otherchronic stressors. FBs were obtained from ALS patients with a variety ofTDP-43 mutations or with repeat expansion in C9orf72, HD patients withhuntingtin (HTT) poly-Q expansion, and Alzheimer's disease patients withPSEN1 or MAPT mutations. By quantitative western blot analysis, STAU1was significantly increased in all patient cell lines (FIGS. 23A-E). TheSTAU1 mRNA levels, however, were unchanged (FIGS. 24A-D).

Stau1 overabundance is associated with autophagy dysfunction. Asdescribed above, in prior studies it was found that STAU1 was primarilydegraded through the autophagosome, presumably owing to its associationwith RNA granules. Further testing was performed here to determinewhether autophagy was impaired in cells with endogenously elevated STAU1and whether exogenous expression of mutant disease-linked proteins couldresult in STAU1 overabundance and impaired autophagy. A master regulatorof autophagy is mTOR and once phosphorylated (P-mTOR), it serves as akinase of several targets including ribosomal protein S6 kinase (S6K1),and eukaryotic initiation factor 4E-binding protein (4E-BP1). FBsobtained from SCA2 patients all showed an increase in total mTOR andactive P-mTOR levels, and an increase in p62 and LC3-II levelsindicating reduced autophagic flux (FIG. 23E). Activation of mTOR wasalso evident in cells from patients with ALS, HD, and AD (FIGS. 23A-D).Increased STAU1 and mTOR levels in these FBs were not the result oftranscriptional changes (FIGS. 24A-D).

It is possible that patient-derived cells may contain other unknownmutations in addition to the respective causative mendelian mutation.Therefore two independent approaches were used to establish a causativerelationship from disease proteins to STAU1 overabundance toautophagosome dysfunction. First, HEK-293 cells were modified using theCRISPR/Cas9 system to introduce disease-causing mutations. When comparedto wild-type HEK-293 cells, cells with endogenous mutated ATXN2,designated ATXN2-Q22/58 KI showed increased STAU1, mTOR, and P-mTORlevels. As expected in the presence of overabundant P-mTOR, downstreameffectors of mTOR signaling showed increased abundance andphosphorylation as well (P-S6 and P-4E-BP1) (FIG. 25A).

Second, we expressed disease causing proteins exogenously in HEK-293cells to determine whether they recapitulated STAU1 overabundance andmTOR activation. This included wild-type or mutant TDP-43 (G298S, A382Tand G348C), C-terminal fragment (CTF) of TDP-43, and MAPT mutation.Exogenous expression of disease-linked proteins resulted in increasedmTOR activities (FIGS. 25B-D). These results support that presence ofmutant disease-linked proteins are directly related to STAU1overabundance and autophagy impairment.

Stau1 is overabundant in mouse models of neurodegeneration. AlthoughSTAU1 overabundance and impaired autophagy were seen in a number of celllines from patients with various neurodegenerative conditions, thisanalysis used cells that are actively dividing. To examine STAU1 levelsin post-mitotic neurons in vivo, brain protein extracts from a number ofmouse models expressing human mutant proteins were evaluated. Aspreviously discussed, STAU1 is overabundant in cerebellar extracts ofSCA2 mice. The state of autophagy was additionally evaluated in thesemice and it was found that the in vivo results mirrored those obtainedusing SCA2 cell lines (FIG. 26A).

The analysis was further extended to a mouse line expressing wild-typeTDP-43 and a mouse line expressing a human BAC with mutant C9orf72repeat. By western blot analysis, both TDP-43^(Tg/+) hemizygous andBAC-C9orf72 mice demonstrated greatly increased Stau1 levels similar tothose seen in ATXN2^(Q127) mice (FIG. 26B-D). Associated with elevatedStau1, it was found that autophagy pathway components were altered,predicting reduced flux by inefficient autophagosome-lysosome fusion.This included total and phosphorylated mTor as well as p62 and LC3-IIwhich functions as a selective autophagy receptor for degradation ofubiquitinated substrates (FIG. 26A-D).

STAU1 directly interacts with the 5′UTR of mTOR and enhances itstranslation. It was further determined if exogenous expression of STAU1in HEK-293 cells resulted in the same alterations that were seen incells with neurodegenerative disease-linked mutations. Expression ofSTAU1 resulted in increased mTOR, P-mTOR, p62, and LC3-II levels (FIG.27A). Levels of mTOR transcripts were unchanged (FIG. 27B). Theseresults indicated that STAU1 overabundance was sufficient to induceautophagic dysfunction.

As STAU1 can enhance translation of mRNAs upon binding to 5′UTRs, it wasfurther tested whether STAU1 was able to bind mTOR RNA and whether thisinteraction would increase translation of mTOR mRNA. Two independentapproaches were used to establish an interaction between STAU1 and mTORRNA. First, Protein-RNA immunoprecipitation (non-RNase A treated)followed by RT-PCR in HEK-293 cells expressing Flag-tagged STAU1demonstrated that Flag-STAU1 pulled down mTOR mRNA but not control GAPDH(FIG. 27C). Second, using bacterially expressed recombinant His-taggedSTAU1 protein and in vitro transcribed DIG-labelled mTOR RNA probes, theinteraction of STAU1 to the mTOR-5′UTR was mapped by northwestern blotanalysis (FIG. 27D-E).

To test the consequence of STAU1 binding to the mTOR RNA, the mTOR-5′UTRwas inserted upstream of luciferase. In the presence of exogenous STAU1,translation was greatly increased (FIG. 27F-H). The specificity of STAU1interaction with the UTR sequence was further tested by substituting themTOR-5′UTR with the PCP2-5′UTR as PCP2 is known only to be subjected toSMD not mediated by its 5′UTR. Exogenous STAU1 resulted in significantinduction of luciferase activity only from mTOR-5′UTR-LUC construct(FIG. 27F-H). The double stranded RNA binding domain 3 (dsRBD3) of STAU1acts as the major functional domain for target RNA interactions. Thetranslational increase was dependent on the presence of this domain asits deletion abrogated the effect of STAU1 on translation (FIG. 27I-K).These data explain mTOR increase and map the interaction to the 5′UTR ofthe mTOR RNA and the dsRBD3 domain of STAU1.

STAU1 responds to a variety of stressors. It was further tested whethersubjecting cells to various forms of stress other than expression ofdisease-linked genes would affect STAU1 and autophagic function.Specifically, HEK-293 cells were subjected to endoplasmic reticulum (ER)stressors (thapsigargin or tunicamycin), a calcium ionophore(ionomycin), oxidative stress (tunicamycin), metal stressor (sodiumarsenite), and temperature stress (hyperthermia) followed by westernblotting. The different forms of stress all induced STAU1 overabundance,mTOR elevation and increased phosphorylation of mTOR targets (FIG.28A-E). To confirm the involvement of STAU1 in the activation of thispathway primary cultures of cortical neurons were established fromwild-type and Stau1^(−/−) mice. For wild-type neurons, treatments withthapsigargin induced Stau1 overabundance and mTor activation withincreased p62 and LC3-II levels. In contrast, Stau1^(−/−) neurons showedminimal response to thapsigargin (FIG. 28F), indicating that theactivation of mTOR and downstream targets is STAU1 dependent. Althoughthe precise signaling pathways are not yet known, these findingsindicate that STAU1 is positioned at the crossroads of several cellularstress pathways.

Reduction of overabundant STAU1 normalizes autophagy. Exogenous STAU1expression was sufficient to change autophagic flux, as shown above.Thus, it was further determined whether STAU1 was necessary. To testthis, STAU1 was knocked down in the presence of mutant disease proteinsin vitro and in vivo. When STAU1 was depleted in ATXN2-Q58 KI cells bySTAU1 RNAi, mTOR levels were normalized. Reduced mTOR activity wasverified by reduced P-mTOR, P-S6, and P-4EBP1 levels, and restoration ofp62 and LC3-II turnover (FIG. 29A). Thus, reducing STAU1 levels wassufficient in rescuing autophagic flux in SCA2 cells.

It was further determined if lowering STAU1 levels could restoreautophagic flux in ALS cellular models. Like in ATXN2-KI cells, loweringSTAU1 levels by RNAi in ALS-TDP-43 or C9orf72 cell models also restoredautophagic flux (FIG. 29B-C). Of note, silencing of STAU1 had no effecton TDP-43 protein levels. These data indicate that STAU1 knockdown maybe useful for preventing maladaptive responses to cellular stress.

Since lowering STAU1 levels can normalize aberrant autophagy associatedwith SCA2, TDP-43, and C9orf72 mutations, it was further tested whetherreducing STAU1 levels is protective of pathological stress.Specifically, HEK-293 cells were treated with STAU1 RNAi followed bywild-type or mutant TDP-43 transfection. In all cases, lowering STAU1normalized mTOR signaling and autophagy (FIG. 30). These resultsestablish that STAU1 plays a key role in protecting cells from a widevariety of pathological stressors.

In vivo results. The availability of a mouse with a Stau1loss-of-function allele allowed the determination of the effect of Stau1reduction using genetic interaction. SCA2 or TDP-43 mouse lines werecrossed with Stau1 KO mice generating animals that had 50% or 0% Stau1protein levels. As described above, it was previously observed thatreduction of Stau1 in vivo improved motor behavior and proteomics inSCA2 mice. mTor, P-mTor, p62, and LC3-II were also measured to determineautophagic flux. Changing Stau1 dosage in wild-type animals did notalter autophagy marker proteins in an appreciable way. In contrast,reducing pathologically elevated Stau1 in SCA2 mice improved markers ofautophagy in a dose-dependent fashion such that levels of all proteinswere returned to near normal (FIG. 31A, B, D). Similar to in SCA2 mice,reducing Stau1 dosage by 50% in TDP-43^(Tg/+) mice resulted in decreasedmTOR activity and restoration of autophagy pathway proteins (FIG. 31C,E).

These results have implications for the basic understanding ofmechanisms regulating autophagy and for identifying novel targets forthe treatment of neurodegeneration. Current understanding of autophagyemphasizes regulation by kinase cascades and by transcriptional changesin key genes involved in autophagy. These results describe an additionalregulatory mechanism that involves RNA-binding proteins, formation ofSGs with the ability of acutely influencing the translation of alreadytranscribed mRNAs. The double-stranded RNA-binding protein STAU1directly interacts with mTOR-mRNA and enhances its translation. As STAU1is itself degraded by autophagy, this establishes an amplificationmechanism for autophagy inhibition. STAU1 is also a key player in RNAdecay, and its overabundance likely affects RNA networks that involvecellular functions and signaling cascades in addition to autophagy.

Example 12—Comparison of the Effect of Various ASOs Targeting Staufen1

The ASOs presented in Table 4 were prepared and evaluated as potentialtherapeutics for targeting Staufen1.

TABLE 4 ASO Sequences Antisense SEQ ID Designation sequence (5′ to 3′)NO: UU00044 TCGGGCTATCATGGCAGTTA  3 UU00045 CTCGGGCTATCATGGCAGTT  4UU00046 TCTCGGGCTATCATGGCAGT  5 UU00047 CTCTCGGGCTATCATGGCAG  6 UU00071GCTGTGGGCGAGGTGCCCCC  7 UU00073 CGGCTGTGGGCGAGGTGCCC  8 UU00181CATGGCAGTTACCGTTGCCT  9 UU00186 GCTATCATGGCAGTTACCGT 10 UU00193AACTCTCGGGCTATCATGGC 11 UU00199 TACAACAACTCTCGGGCTAT 12 UU00223TAAACTCTGGCTGCTGCTCC 13 UU00224 AGTGGTTAATAAACTCTGGC 14 UU00225GTTCTGAGAGGTTAAGTGGT 15 UU00226 TTTGTTCAGTTCTGAGAGGT 16 UU00227TTCCAGGAACAATGTTGTCT 17 UU00229 AAACAGCTTTCAGTGCAGGT 18 UU00230ACAAATGCAGGTAAACAGCT 19 UU00231 AGACATGGTCACTTTCAACA 20 UU00232CACTTGAACTTGAGACATGG 21 UU00233 TTCTGAACTTGCACTTGAAC 22 UU00236TCAGTATTTGGCTCCCTGAG 23 UU00237 TCTTGTTCAGTATTTGGCTC 24 UU00239GCTGTGAGAGAAGAGACTGG 25 UU00243 GGTCTACCTGCATTTTCAGA 26 UU00245AGCTGCACTGGTGGATGTAA 27 UU00246 CACAGTGCATTTAGTTCTAC 28 UU00247TCATGCACAGTGCATTTAGT 29 UU00248 CCAAGTTTCATGCACAGTGC 30 UU00249CAACAGGCTTATACATTGGT 31 UU00250 AGTTATAGGTGGACTGCATC 32 UU00254CCCACAGAAAGTTCCACTTG 33 UU00256 TGCCATTAAATTGCTGTCCT 34 UU00257GTCTTGTCTTTCCTTTGCCA 35 UU00258 GCAGCCTGTCTTGTCTTTCC 36 UU00261GTGCAATCTCAAACACTTGA 37 UU00263 TGGTCACAAAGTTCTTCATG 38 UU00291TGGTGTGATGTCCTTGACTA 39 UU00307 GGTTCAGCACCTCCCACACA 40

Mouse NIH3T3 cells were transfected with 250 nM of the ASOs indicated inTable 4 or phosphate buffered saline as a negative control. 4 daysfollowing the transfection, Stau1 expression was determined byquantitative PCR using primers targeting exon 4. All staufen ASOs targetboth human and mouse staufen1 sequences and had the 5-10-5 MOE gapmerchemistry. Results of some ASOs are presented in FIG. XX. Additionally,Stau1 expression was determined by western blotting. Some of the westernblotting results are presented in FIG. 32A.

SCA2 patient derived skin cell fibroblasts (SCA2-03[Q22/Q35]) were alsotransfected with either 200 nM or 300 nM amounts of various ASOs orphosphate buffered saline. After 4 days STAU1 expression was determinedby quantitative PCR. Some results are presented in FIG. 32B.

Further, TDP-43+/− transgenic mice were treated byintracerebroventricular injection with 250 mg of various ASOs or saline.After 15 days treatment the spinal cords were removed and Stau1expression was determined by quantitative PCR (FIG. 32C). Microgliosiswas also assessed by determining Aif1 expression, revealing no increasedAif1 expression compared to the control, or no evidence for microgliosis(FIGS. 32D-E). Values are means and standard deviations. N number micewere 5 (PBS), 4 (UU000181), 1 (UU00186), 2 (UU00045), 4 (UU00193), 5(UU00199).

It should be understood that the above-described methods are onlyillustrative of some embodiments of the present invention. Numerousmodifications and alternative arrangements may be devised by thoseskilled in the art without departing from the spirit and scope of thepresent invention and the appended claims are intended to cover suchmodifications and arrangements. Thus, while the present invention hasbeen described above with particularity and detail in connection withwhat is presently deemed to be the most practical and preferredembodiments of the invention, it will be apparent to those of ordinaryskill in the art that variations including, may be made withoutdeparting from the principles and concepts set forth herein.

1-49. (canceled)
 50. A therapeutic composition comprising: an amount ofa Staufen1-regulating agent sufficient to treat a neurological conditionby reducing Staufen1 in a target cell by at least 30% when administeredin an effective dosing regimen as compared to levels of Staufen1 priorto or without administration of the Staufen1-regulating agent; and apharmaceutically acceptable carrier.
 51. The composition of claim 50,wherein the Staufen1-regulating agent comprises an antisenseoligonucleotide (ASO), a small interfering RNA (siRNA), a smallmolecule, a micro RNA (miRNA), a ribozyme, or a combination thereof. 52.The composition of claim 50, wherein the Staufen1-regulating agentreduces Staufen1 expression, reduces Staufen1 activity, reduces orblocks Staufen1 interaction with mRNAs resulting in altered mRNAexpression or abundance, or a combination thereof.
 53. The compositionof claim 50, wherein the Staufen1-regulating agent comprises anucleotide sequence that is at least 80% homologous to the 10 middlenucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO:
 20. SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or acombination thereof.
 54. The composition of claim 50, wherein theStaufen1-regulating agent is present in the formulation at aconcentration of from about 1 picomolar (pM) to about 100 millimolar(mM).
 55. The composition of claim 50, wherein the Staufen1-regulatingagent is present in the formulation at a concentration of from about0.001 μg/g to about 200 mg/g.
 56. The composition of claim 50, furthercomprising a delivery vector to facilitate delivery of theStaufen1-regulating agent into a target cell.
 57. The composition ofclaim 56, wherein the delivery vector is a viral vector.
 58. Thecomposition of claim 57, wherein the viral vector is a member of thegroup consisting of: a retrovirus, a lentivirus, a cytomegalovirus, anadenovirus, an adeno-associated virus, and combinations thereof.
 59. Thecomposition of claim 56, wherein the delivery vector is a non-viralcarrier selected from the group consisting of: an aptamer, anantibodies, an antibody fragment, a polypeptides, N-acetylgalactosamine,a vitamin, a small organic molecules, a polycationic peptide, apolymers, a dendrimer, a lipid, a lysosomal carrier, a liposome, amicelle, a quantum dot, a nanoparticle, and combinations thereof. 60.The composition of claim 50, wherein the pharmaceutically acceptablecarrier is formulated for administration via injection, enteraladministration, transdermal administration, transmucosal administration,inhalation, or implantation.
 61. The composition of claim 50, whereinthe pharmaceutically acceptable carrier comprises water, a solubilizingagent, a tonicity agent, a pH adjuster, a buffering agent, apreservative, a chelating agent, a bulking agent, a binder, adisintegrant, a filler, a thickener, a dispersant, an emulsifier, anemollient, or combinations thereof.
 62. The composition of claim 50,further comprising a supplementary therapeutic agent.
 63. Thecomposition of claim 62, wherein the supplementary therapeutic agent isa member selected from the group consisting of: a dopaminergic agent, acholinesterase inhibitor, an antipsychotic agent, an analgesic, ananti-inflammatory agent, an inducer of autophagy, and combinationsthereof.
 64. A method of treating a neurological condition associatedwith a mutation of the C9orf72 gene, the TDP-43 gene, the HTT gene, thePSEN1 gene, the MAPT gene, or a combination thereof, comprising:introducing an amount of a Staufen1-regulating agent to a target cellthat is sufficient to minimize Staufen1-induced dysregulation of RNAmetabolism.
 65. The method of claim 64, wherein introducing comprisesintroducing the Staufen1-regulating agent to the target cell with anon-viral vector.
 66. The method of claim 64, wherein introducingcomprises introducing the Staufen1-regulating agent to the target cellwith a viral vector.
 67. The method of claim 64, wherein introducingcomprises introducing the Staufen1-regulating agent without a deliveryvector.
 68. The method of claim 64, wherein the amount of Staufen1-regulating agent increases PCP2 mRNA levels in the target cell, CALB1mRNA levels in the target cell, or both as compared to said levels priorto or without introducing the Staufen1-regulating agent.
 69. The methodof claim 64, wherein the amount of Staufen1-regulating agent reducesStaufen1 levels in the target cell by at least 30% as compared toStaufen1 levels in the target cell prior to or without introduction ofthe Staufen1-regulating agent.
 70. The method of claim 64, wherein theStaufen1-regulating agent reduces Staufen1 expression, reduces Staufen1activity, reduces or blocks Staufen1 interaction with mRNAs resulting inaltered mRNA expression or abundance, or a combination thereof.
 71. Themethod claim 64, wherein the Staufen1-regulating agent comprises anantisense oligonucleotide (ASO), a small interfering RNA (siRNA), asmall molecule, a micro RNA (miRNA), a ribozyme, or a combinationthereof.
 72. The method of claim 64, wherein the Staufen1-regulatingagent comprises a nucleotide sequence that is at least 80% homologous tothe 10 middle nucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO:
 20. SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQID NO: 42, or a combination thereof.